Methods and microfluidic devices for the manipulation of droplets in high fidelity polynucleotide assembly

ABSTRACT

Methods and devices are provided for manipulating droplets on a support using surface tension properties, moving the droplets along a predetermined path and merging two droplets together enabling a number of chemical reactions. Disclosed are methods for controlling the droplets volumes. Disclosed are methods and devices for synthesizing at least one oligonucleotide having a predefined sequence. Disclosed are methods and devices for synthesizing and/or assembling at least one polynucleotide product having a predefined sequence from a plurality of different oligonucleotides having a predefined sequence. In exemplary embodiments, the methods involve synthesis and/or amplification of different oligonucleotides immobilized on a solid support, release of synthesized/amplified oligonucleotides in solution to form droplets, recognition and removal of error-containing oligonu-cleotides, moving or combining two droplets to allow hybridization and/or ligation between two different oligonucleotides, and further chain extension reaction following hybridization and/or ligation to hierarchically generate desired length of polynucleotide products.

RELATED APPLICATIONS

This application claims the benefit from U.S. provisional applicationSer. No. 61/257,591, filed Nov. 3, 2009, U.S. provisional applicationSer. No. 61/264,641, filed Nov. 25, 2009, U.S. provisional applicationSer. No. 61/310,069, filed Mar. 3, 2010, the entire contents of all ofwhich are herein incorporated by reference.

FIELD OF THE INVENTION

Methods and devices provided herein generally relate to droplet-basedliquid manipulation on a substrate. In some embodiments, picoliter andsub-picoliter volume dispensing and droplet translocation technologiesare applied to access and manipulate the oligonucleotides on asubstrate. More particularly, methods and devices herein relate to theassembly of high fidelity nucleic acids having a predefined sequence andlibraries of such predefined nucleic acids, the methods and devicesusing microvolume reactions, error filtration, hierarchical assembly,and/or sequence verification on a solid support.

BACKGROUND

Using the techniques of recombinant DNA chemistry, it is now common forDNA sequences to be replicated and amplified from nature and thendisassembled into component parts. As component parts, the sequences arethen recombined or reassembled into new DNA sequences. However, relianceon naturally available sequences significantly limits the possibilitiesthat may be explored by researchers. While it is now possible for shortDNA sequences to be directly synthesized from individual nucleosides, ithas been generally impractical to directly construct large segments orassemblies of polynucleotides, i.e., polynucleotide sequences longerthan about 400 base pairs. Furthermore, the error rate ofchemically-synthesized oligonucleotides (deletions at a rate of 1 in 100bases and mismatches and insertions at about 1 in 400 bases) exceeds theerror rate obtainable through enzymatic means of replicating an existingnucleic acid (e.g., PCR). Therefore, there is an urgent need for newtechnology to produce high-fidelity polynucleotides.

Oligonucleotide synthesis can be performed through massively parallelcustom syntheses on microchips (Zhou et al. (2004) Nucleic Acids Res.32:5409; Fodor et al. (1991) Science 251:767). However, currentmicrochips have very low surface areas and hence only small amounts ofoligonucleotides can be produced. When released into solution, theoligonucleotides are present at picomolar or lower concentrations persequence, concentrations that are insufficiently high to drivebimolecular priming reactions efficiently. Current methods forassembling small numbers of variant nucleic acids cannot be scaled up ina cost-effective manner to generate large numbers of specified variants.As such, a need remains for improved methods and devices forhigh-fidelity gene assembly and the like.

SUMMARY

Methods and devices provided herein generally relate to droplet-basedliquid manipulation on a substrate. In some embodiments, picoliter andsub-picoliter volume dispensing and droplet surface tensions are used toaccess and manipulate the droplets on a substrate. It is an object ofthis invention to provide microfluidic devices for the manipulations ofdroplets. It is also an object of the invention to provide methods anddevices for processing nucleic acids, (e.g. oligonucleotides)amplification reactions and assembly reactions.

Aspects of the invention relate to methods and devices for preparingoligonucleotides on a support. In some embodiments, a support comprisinga plurality of surface-bound single stranded oligonucleotides which arecontained within one or more droplets of a predefined volume of solutionis provided. In some embodiments, a plurality of complementaryoligonucleotides is generated within the droplet volume by exposing theplurality of surface-bound oligonucleotides to conditions suitable for atemplate-dependent synthesis and the volume of the one or more dropletsof solution is adjusted or maintained. In some embodiments, the volumeof the droplets of solution is maintained by maintaining the dropletsunder conditions that substantially limit water evaporation. Forexample, the plurality of surface-bound oligonucleotides may be coupledto the surface at a feature that is selectively coated with a coatingmaterial. The coating material may have water trapping properties andmay be selected from the group of colloidal silica, peptide gel,agarose, solgel and polydimethylsiloxane, or any combination thereof. Inanother embodiment, water evaporation is limited by blocking theinterface of the droplet with the atmosphere. For example, the dropletscan be overlaid with a non-miscible liquid thereby preventing waterevaporation of the solution. In some embodiments, the non-miscibleliquid forms a lipid bilayer and the lipid bilayer is evaporated to forma thin film at the surface of the droplet. In some exemplaryembodiments, the non-miscible liquid is a solvent or mineral oil. Thenon-miscible liquid may spotted onto the droplet, for example using aninkjet or mechanical device. In some embodiments, the volume of thedroplet is maintained by adjusting the droplet volume by providingadditional solution in response to evaporation. The water addition canbe semi-continuous and can be added using an inkjet device. In someembodiments, water evaporation is limited by controlling the localhumidity around the droplets. For example, the local humidity isincreased by depositing satellite droplets in the vicinity of thedroplets.

Aspects of the invention relate to methods and devices for monitoring aplurality of isolated reaction volumes on a support. In someembodiments, the method comprises providing a first support comprising aplurality isolated reaction volumes having a predefinedsurface-to-volume ratio; providing a second support comprising at leastone monitoring isolated volume, wherein the monitoring volume has anidentical surface-to-volume ratio to at least one of the reactionvolume; and monitoring the volume of the at least one monitoringisolated volume, wherein the modification of the isolated monitoringvolume is indicative of the modification of at least one isolatedreaction volume. The first and second support can be the same or theisolated reaction volumes and isolated monitoring volumes are depositedon the same support. In preferred embodiments, the isolated volumes aredroplets. In preferred embodiments, the isolated reaction volumecomprises a solvent and wherein the monitoring volume comprises the samesolvent. The reaction volume can comprise oligonucleotides. Modificationof volume, such as increase of volume or decrease of volume ismonitored. The reaction volumes and the isolated monitoring volumes aresubjected to preselected conditions such as temperature, pressure, andgas mixture environment. The surfaces of the isolated reaction volumesand the isolated monitoring volumes are in contact with the preselectedgas mixture, for example a gas mixture having predefined molar ratio ofsolvent vapor and carrier gas. In some embodiments, the conditions aremodified to induce isolated volume growth. Yet in other embodiments, theconditions are modified to induce isolated volume evaporation. In someembodiments, the monitoring isolated volumes are placed on a mirrorsurface and the volume of the at least one monitoring isolated volume ismonitored using an optical system. In an exemplary embodiment, thevolume of the at least one monitoring isolated volume is monitored bymeasuring the intensity of an optical beam reflected on the secondsupport. In some embodiments, the volume changes of the isolatedreaction volumes is measured by providing a second support, such as amirror and measuring the condensation on the second support using anoptical system. In preferred embodiments, the condensation is measuredby measuring the intensity of an optical beam reflected on the mirror.

In some embodiments, the surface bound oligonucleotides comprise aprimer binding site and the solution comprises a polymerase, at leastone primer and dNTPs wherein the primer is complementary to the primerbinding site. The primer may be a unique primer, a universal primer, apair of primers, a pair of unique primers, or a pair of universalprimers. Oligonucleotides may be amplified by subjecting the pluralityof surface-bound oligonucleotides to thermocycling thereby promotingprimer extension.

In some embodiments, methods and devices for moving a droplet on asubstrate are provided. In some embodiments, the substrate may bepartitioned with a plurality of surface modifiers. The substrate may bepartitioned according to a pattern such as an alternative pattern. Theplurality of modifiers may comprise a plurality of first modifiers andplurality of second modifiers. In some embodiments, the plurality offirst modifiers has a contact angle smaller than the plurality of secondmodifiers. In some embodiments, each of the plurality of first modifiersis associated with a different contact angle and the plurality of firstmodifiers is arranged in a series of decreasing or increasing contactangles. The plurality of modifiers can then form a hydrophilic gradient.In some embodiments, the first modifier is contacted with a droplet, andthe droplet is moved on a substrate along a path towards the firstmodifiers having smaller contact angles. In an exemplary embodiment, thefirst modifier is contacted using an inkjet device. Therefore, thedroplet may be moved along a hydrophilic gradient. In some embodiments,the first and second modifiers' contact angles differ by more than 30°.In some embodiments, the first modifiers comprise a plurality ofoligonucleotides. In some embodiment, the second modifier correspond tothe unmodified substrate surface. In another embodiment, the secondmodifier comprises a different surface modifier than the first modifier.In some embodiments, the first modifier is surrounded by secondmodifiers. In some embodiment, the droplet moves along a pre-determinedpath comprising a pattern of modifiers. In preferred embodiments, thepath is a hydrophilic gradient and the droplet move according tosurface-tension properties. Droplets may be moved along a one or a twodimensional path.

Aspects of the invention relate to methods and devices for moving andmerging droplets on substrate surface comprising a plurality offeatures. In some embodiments, a first feature is contacted with a firstdroplet, and a second feature is contacted with a second droplet. Thefirst and second features may be adjacent to each others. In preferredembodiment, the second droplet volume is greater than the first dropletvolume. The first of the second droplet is then contacted with a thirddroplet volume allowing the merging of the droplets into a fourthdroplet. In some embodiments, the volume of the first droplet istherefore moved from the first feature to the second feature on thesubstrate. Volume of the resultant droplet may be reduced to theoriginal volume of the first droplet and steps may be repeated. Usingthe process of adjusting volumes and using surface tension properties,droplets can be moved along a predetermined path.

Other aspects of the invention relate to methods and devices forconducting sub-microvolume specified reactions within a droplet. In someembodiments, a substrate is provided comprising a plurality ofsurface-bound single-stranded oligonucleotides at discrete features. Inother embodiments, only a selected set of oligonucleotides suitable forhydration are hydrated while the remainder of the support remains dry.In one embodiment, each oligonucleotide has a predefined sequencedifferent from the predefined sequence of the oligonucleotide bound to adifferent feature. In some embodiments, a set of predefined features maybe selectively hydrated, thereby providing hydrated oligonucleotides. Inanother embodiment, the hydrated oligonucleotides are exposed to furtherprocessing within a droplet volume. In some embodiments, theoligonucleotides are subjected to amplification. As each feature canselectively be hydrated, amplification will take place only at specificfeatures comprising a droplet. In some embodiments, the droplet acts asa virtual reaction chamber. In some embodiments, the features can behydrated with a solution promoting primer extension onto at least onefeature creating at least one first stage droplet. For example, thesolution may comprise a polymerase, at least one primer and dNTPswherein the primer is complementary to a primer binding site. The primermay be a unique primer, a universal primer, a pair of primers, a pair ofunique primers, or a pair of universal primers. Oligonucleotides may beamplified by subjecting at least one feature to thermocycling therebypromoting primer extension. In some other embodiments, the entiresurface is subjected to thermocycling. In subsequent steps, the surfaceis heated to a denaturing temperature thereby providing a plurality ofsingle-stranded complementary oligonucleotides within the first stagedroplet. In other embodiments, the water evaporation or volume of thedroplets is controlled. For example, the discrete features may beselectively coated with a coating material which may have water-trappingproperties. In some embodiments, the coating material may be colloidalsilica, peptide gel, agarose, solgel, polydimethylsiloxane, or anycombination thereof. In another embodiment, water evaporation is limitedby blocking the interface of the droplet with the atmosphere. Forexample, the droplets can be overlaid with a non-miscible liquid therebypreventing water evaporation of the solution. In some embodiments, thenon-miscible liquid forms a lipid bilayer and the lipid bilayer isevaporated to form a thin film at the surface of the droplet. In someexemplary embodiments, the non-miscible liquid is a solvent or mineraloil. The non-miscible liquid may spotted onto the droplet, for exampleusing an inkjet or mechanical device. In some embodiments, the volume ofthe droplet is maintained by addition of solution to the droplet. Thewater addition can be semi-continuous and can be added using an inkjetdevice. In some embodiments, water evaporation is limited by controllingthe local humidity around the droplets. For example, the local humidityis increased by depositing satellite droplets in the vicinity of thedroplets.

Aspects of the invention also relate to methods and devices for removingerror-containing oligonucleotides from a plurality of amplifiedoligonucleotides. In some embodiments, the method comprises the steps ofhydrating at least one first feature of the solid support following theamplification step forming a droplet comprising oligonucleotidesduplexes; heating the solid support to a first melting temperature understringent melt conditions, thereby denaturing duplexes comprisingerror-containing oligonucleotides and releasing error-containingoligonucleotides; removing the error-containing oligonucleotides fromthe solid support; optionally repeating previous steps on at least onesecond different feature and at least one different melting temperature;denaturing error-free duplexes; and releasing error-freeoligonucleotides in solution. Stringent melt conditions can bedetermined by a real-time melt curve. In some embodiments, the supportis dried prior to first and subsequent hydrating step. In someembodiments, a subset of discrete features is selectively heated. Forexample, one or more discrete features are selectively heated using adigital mirror device.

Other aspects of the invention relate to methods and devices forassembling at least one polynucleotide having a predefined sequence on asupport. In one embodiment, a support is provided that comprises atleast one feature having a plurality of surface-bound single-strandedoligonucleotides that are in a dry form and suitable for hydration. Eachplurality of oligonucleotides is bound to a discrete feature of thesupport, and the predefined sequence of each plurality ofoligonucleotides attached to the feature is different from thepredefined sequence of the plurality of oligonucleotides attached to adifferent feature. At least one feature is hydrated thereby providinghydrated oligonucleotides within a droplet. In some embodiments, atleast one plurality of oligonucleotides is synthesized in a chainextension reaction on a first feature of the support bytemplate-dependent synthesis. The products of chain extension aresubjected to at least one round of denaturation and annealing. Thesupport is then heated to a first melting temperature under stringentmelt conditions thereby denaturing duplexes comprising error-containingoligonucleotides and releasing error-containing oligonucleotides insolution. Error-containing oligonucleotides are removed from thesupport. The steps can be repeated on at least one other feature and atleast one different melting temperature. Error-free duplexes aredenatured and error-free oligonucleotides are released in solutionwithin a first stage droplet. A first droplet comprising a firstplurality of substantially error-free oligonucleotides can then becombined to a second droplet comprising a second plurality ofsubstantially error-free oligonucleotides, wherein a terminal region ofthe second plurality of oligonucleotides comprise complementarysequences with a terminal region of the first set of plurality ofoligonucleotides. The first and second plurality of oligonucleotides canthen be contacted under conditions that allow one or more of annealing,chain extension, and denaturing. In some embodiments, the first andsecond droplets are combined by merging the droplets into a second stagedroplet. First and/or second droplets can be moved from a first featureto a second feature of the support. In some embodiments, the surface iscoated with a low melting-point substance for storage, for example wax,for storage. In some embodiments, the reactions are initiated by heatingthe surface above the low-melting point. Yet in other embodiments, thereactions are initiated by hydrating the discrete features.

Aspect of the invention relate to methods and devices of preparing aplurality of oligonucleotides using two supports. In some embodiments, afirst support comprising a plurality of discrete features is providedand a second support is provided, the second support comprising an arrayof electrodes. The first and second support may be the same. Eachfeature comprising a plurality of surface-bound single-strandedoligonucleotides having a predefined sequence. In some embodiments, adroplet is dispensed at a first selected feature and at least oneplurality of oligonucleotides is synthesized in a chain extensionreaction on the first feature of the support by template-dependentsynthesis. The products of chain extension are then subjected to atleast on round of denaturation and annealing to for duplexoligonucleotides and the duplexes are exposed to conditions promotingerror reduction. Error reduction may be an error filtration process, anerror correction process or an error neutralization process. In someembodiments, the error reduction utilizes a mismatch endonuclease suchas a CELL or a Surveyor™ endonuclease to cleave heteroduplexes. In someembodiments, the droplet is moved to the selected feature by activatingand deactivating a selected set of electrodes. In some embodiments, thetwo supports are arranged together relative to each other by a distancesufficient to define a space in some embodiments, the error containingduplexes are exposed with a mismatch endonuclease under conditions thatpermit cleavage of oligonucleotide duplexes having at least one mismatchand the cleaved duplexes are removed. In some embodiments, the methodfurther comprises denaturing surface-bound cleaved duplexes, removingsingle stranded cleaved oligonucleotides, denaturing surface-boundsubstantially error free oligonucleotide duplexes and releasing a firstplurality of substantially error-free complementary oligonucleotides infirst droplet volume. In some embodiments, a second plurality ofsubstantially error-free oligonucleotides is released in second dropletvolume. The first and second droplet may be moved towards a thirdfeature to form a merged droplet by activating and deactivating a set ofelectrodes, thereby mixing the first and second droplets compositiontogether. In some embodiments, the method further combines a firstdroplet comprising a first plurality of substantially error-freeoligonucleotides to a second droplet comprising a second plurality ofsubstantially error-free oligonucleotides, wherein a terminal region ofthe second plurality of oligonucleotides comprises complementarysequences with a terminal region of the first set of plurality ofoligonucleotides. The first and second plurality of oligonucleotides arethen contacted under conditions that allow one or more of annealing,chain extension and denaturing reaction. In some embodiments, one ormore discrete features are selectively heated, for example, using adigital mirror device.

In other embodiments, a plurality of oligonucleotides having apredefined sequence are synthesized on a support. First, a plurality ofsurface-bound single-stranded oligonucleotides having a predefinedsequence are provided wherein the plurality of oligonucleotides aresuitable for hydration and wherein each plurality of oligonucleotides isbound to a discrete feature of the support, wherein the predefinedsequence of each plurality of oligonucleotides attached to the featureis different from the predefined sequence of the plurality ofoligonucleotides attached to a different feature. One feature isselectively inactivated by overlaying the selected feature with animmiscible solution and at least one second feature is selectivelyhydrated thereby providing hydrated oligonucleotides. At least oneplurality of oligonucleotides is then synthesized in a chain extensionreaction on a second feature of the support by template-dependentsynthesis. The oligonucleotide duplexes are subjected to error-reductionand substantially error-free complementary oligonucleotides are releasedin a droplet volume. In some embodiments, an inactivated first featureis activated by removing the immiscible solution such as oil. In someembodiments, the method further comprises selectively hydrating thefirst feature thereby providing hydrated oligonucleotides, synthesizinga plurality of oligonucleotides in a chain extension reaction on a firstfeature of the support by template-dependent synthesis, subjectingoligonucleotide duplexes to error-reduction, and releasing substantiallyerror-free complementary oligonucleotides in a droplet volume. In someembodiments, the droplets may be moved by electrowetting.

In some embodiments, the plurality of single-stranded oligonucleotidesare synthesized at each feature using high-voltage complementarysemiconductor device. In other embodiments, the plurality ofsingle-stranded oligonucleotides are synthesized at each feature usingemulsion droplets.

Aspects of the invention relates to methods and devices for thesynthesis of at least one oligonucleotide of a predefined sequence ontoa discrete feature of the support. In some embodiments, a first supportcomprising a plurality of discrete features and a second supportcomprising a high density array of electrodes are provided. Droplets areprovided on selected features, the droplets comprising a reagent forperforming a step of oligonucleotide synthesis. Preferably, the dropletsare moved using high voltage electronics to a second selected featurefor performing a step of the oligonucleotide synthesis, therebyproducing the oligonucleotide of interest. In other embodiments, asupport is provided, the support comprising a plurality of discretefeatures. Emulsion droplets are provided onto selected features, thedroplet comprising a reagent for performing a step of oligonucleotidesynthesis. Droplets containing different reagents for performing a stepof oligonucleotide synthesis can be merged to perform and synthesize theoligonucleotide of interest. The reagents may be selected from the groupconsisting of a A coupling reagent, T coupling reagent, C couplingreagent, G coupling reagent, U coupling reagent, deblocking reagent,oxidation reagent, capping reagent. In some embodiments, each dropletcomprises a reagent for the oligonucleotide synthesis, each reagentbeing encapsulated into an aqueous droplet within an immiscible compoundsuch as oil and surfactant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates an embodiment of the device comprising a substrate(1) with a surface (2), the surface being partitioned with modifiers (3)or (3) and (4).

FIG. 1B illustrates an embodiment of the device comprising partitioningof surface (2) with modifiers (3).

FIG. 1C illustrates an embodiment of the device comprising possiblepatterning configurations.

FIG. 2A illustrates a non-limiting example of droplet footprint on asubstrate (1) comprising a surface (2) and modifiers (3), the contactangle of the droplet being smaller on (2) than on (3).

FIG. 2B illustrates a non-limiting example of the merging of twodroplets (21 and 22) separated by a distance (29) using a merger droplet(23). FIG. 2B illustrates a non-limiting example of possible footprint(24A; 24B, 24C) of the merged droplet (24).

FIG. 3 illustrates a non-limiting embodiment of the merging and the moveof droplet 31 with droplet 32 using a merger droplet 33 and theresultant merger droplet footprint 34A and 34B.

FIG. 4A illustrates a non-limiting embodiment of the merging of asmaller droplet 41 with a larger droplet 42, using a merger droplet 43,into a merged droplet 44A and the repeated steps to allow the move ofthe droplet to a selected location of the substrate.

FIG. 4B illustrates a non limiting embodiment of droplet displacement.

FIG. 5A illustrates a non-limiting embodiment of a droplet movementalong a hydrophilic substrate 51 from a more hydrophobic location 52 toa more hydrophilic location 53 and then 54.

FIG. 5B illustrates a non-limiting embodiment of a droplet movementalong a hydrophilic substrate 51 from a more hydrophobic location 52 toa more hydrophilic location 53 and then 54 on a support comprisingbends.

FIG. 5C illustrates a non-limiting embodiment of a droplet movementalong a hydrophilic substrate 51 from a more hydrophobic location 52 toa more hydrophilic location 53 and then 54 on a support comprisingvarying widths.

FIG. 6 illustrates a non-limiting example showing graphically withvarying gray scale how the “solution” droplet is diluted by the “merge”droplet and the “anchor” droplet and is then re-concentrated byevaporation.

FIG. 7A illustrates an embodiment of the device comprising a source wellplate (101), containing the reagents for reactions, a solid support(102), with solid-surface attached or supported molecules, a transducer(103), a coupling fluid (104), reagents (105) inside a well, solidattached or surface supported molecules (110), surface droplets (111)formed by the dispensed droplets (106), a “merge” droplet (112)dispensed between two surface droplets (111), a surface droplet (170)dispensed for the purpose of alignment of the droplet to the solidattached molecules (111), an electronics camera (171) and used toprovide physical registration (positioning) and a surface mark (172)fixed on the solid support (102).

FIG. 7B illustrates an embodiment using an inkjet device for dropletdispensing. The head assembly (180) includes multiple jetting modules(181), with each module containing at least one reservoir (183) havingat least one inlet (184). Each jetting module can have one or more thanone nozzle (182), which can be arranged in a 1D or 2D array to form anozzle pattern. The nozzles can have well defined dimensions to allowdroplets (106) to form under the influence of a mechanical wavegenerated by a transducer (185).

FIG. 8 illustrates an embodiment of thermal control device and procedurecomprising solid support substrate (401) comprising immobilizedmolecules (404); an optical absorbent material (402) in the surfacedroplet (403), the surface droplet comprising molecules (409) insolution, an optical absorbent material (405) on the surface of 401, anoptical energy source (406), a scanning setup (407), energy beams (408)and a plurality of reaction sites (420, 421, 422, 423).

FIG. 9 illustrates an exemplary method of error filtration.

FIG. 10 illustrates a non-limiting example of selective error-removal oferror-containing oligonucleotides.

FIG. 11A illustrates an embodiment of a solid support comprisingdifferent molecules (A, B, C, etc.) and a non-limiting example of anassembly strategy.

FIG. 11B illustrates a non-limiting example of an assembly strategy.

FIG. 12 illustrates an embodiment of a solid support comprisingdifferent and unique molecules (201, 202, 203, 204) supported orattached to the surface of 102, a unique molecule (250) supported orattached to the surface of 102 at multiple positions and other uniquemolecules (299) supported or attached to the surface of 102.

FIG. 13A illustrates a non limiting embodiment of hierarchical assemblyof S1, S2, . . . Sn group members to form S1−n.

FIG. 13B illustrates a non-limiting embodiment of hierarchical assemblyof four assembly sub-groups members M, Q, R, S.

FIG. 14 illustrates a non-limiting embodiment of the wash intransportation process.

FIG. 15 illustrates a cross section of an electrowetting based dropletmicrofluidic substrate.

FIG. 15A illustrates a cross section of the electrowetting device with atop and a bottom side electrode.

FIG. 15 B illustrates a configuration of an electrowetting device withonly a top side electrode.

FIG. 15C illustrates a configuration of the device with only a bottomside electrode

FIG. 16 illustrates a non limiting embodiment of the electrowettingdevice with a top side electrode.

FIG. 17 illustrates a non limiting embodiment of the electrowettingdevice with a bottom side electrode.

FIG. 18 illustrates a non limiting embodiment of polymer assembly.

FIG. 19 illustrates a non limiting embodiment of selective error-removalof error-containing oligonucleotides.

FIG. 20 illustrates a non limiting embodiment of selective error-removalof error-containing oligonucleotides.

FIG. 21 illustrates a non limiting embodiment of polynucleotide assemblyusing an electrowetting device.

FIG. 22 illustrates an exemplary method using an immiscible fluid systemin polynucleotide assembly.

FIG. 23 illustrate non-limiting embodiments of a microvolume sealedplate device to control humidity.

FIG. 24 illustrate non-limiting embodiments of a feedback controlledhumidity chamber.

FIG. 25 illustrates a non-limiting embodiment of a control loop humiditysystem.

FIG. 26 illustrates a non-limiting embodiment of plate to platetransfers.

FIG. 27 illustrates a non-limiting embodiment of plate to platetransfers.

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are microfluidic devices for the manipulation ofdroplets on a substrate. Methods and devices for synthesizing oramplifying oligonucleotides as well for preparing or assembling longpolynucleotides having a predefined sequence are provided herein.Aspects of the technology provided herein are useful for increasing theaccuracy, yield, throughput, and/or cost efficiency of nucleic acidsynthesis and assembly reactions. As used herein the terms “nucleicacid”, “polynucleotide”, “oligonucleotide” are used interchangeably andrefer to naturally-occurring or synthetic polymeric forms ofnucleotides. The oligonucleotides and nucleic acid molecules of thepresent invention may be formed from naturally occurring nucleotides,for example forming deoxyribonucleic acid (DNA) or ribonucleic acid(RNA) molecules. Alternatively, the naturally occurring oligonucleotidesmay include structural modifications to alter their properties, such asin peptide nucleic acids (PNA) or in locked nucleic acids (LNA). Thesolid phase synthesis of oligonucleotides and nucleic acid moleculeswith naturally occurring or artificial bases is well known in the art.The terms should be understood to include equivalents, analogs of eitherRNA or DNA made from nucleotide analogs and as applicable to theembodiment being described, single-stranded or double-strandedpolynucleotides. Nucleotides useful in the invention include, forexample, naturally-occurring nucleotides (for example, ribonucleotidesor deoxyribonucleotides), or natural or synthetic modifications ofnucleotides, or artificial bases. As used herein, the term monomerrefers to a member of a set of small molecules which are and can bejoined together to form an oligomer, a polymer or a compound composed oftwo or more members. The particular ordering of monomers within apolymer is referred to herein as the “sequence” of the polymer. The setof monomers includes but is not limited to, for example, the set ofcommon L-amino acids, the set of D-amino acids, the set of syntheticand/or natural amino acids, the set of nucleotides and the set ofpentoses and hexoses. Aspects of the invention described hereinprimarily with regard to the preparation of oligonucleotides, but couldreadily be applied in the preparation of other polymers such as peptidesor polypeptides, polysaccharides, phospholipids, heteropolymers,polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines,polyarylene sulfides, polysiloxanes, polyimides, polyacetates, or anyother other polymers.

As used herein, the term “predefined sequence” means that the sequenceof the polymer is known and chosen before synthesis or assembly of thepolymer. In particular, aspects of the invention is described hereinprimarily with regard to the preparation of nucleic acids molecules, thesequence of the oligonucleotides or polynucleotides being known andchosen before the synthesis or assembly of the nucleic acid molecules.In some embodiments of the technology provided herein, immobilizedoligonucleotides or polynucleotides are used as a source of material. Invarious embodiments, the methods described herein use oligonucleotides,their sequence being determined based on the sequence of the finalpolynucleotides constructs to be synthesized. In one embodiment,“oligonucleotides” are short nucleic acid molecules. For example,oligonucleotides may be from 10 to about 300 nucleotides, from 20 toabout 400 nucleotides, from 30 to about 500 nucleotides, from 40 toabout 600 nucleotides, or more than about 600 nucleotides long. However,shorter or longer oligonucleotides may be used. Oligonucleotides may bedesigned to have different length. In some embodiments, the sequence ofthe polynucleotide construct may be divided up into a plurality ofshorter sequences that can be synthesized in parallel and assembled intoa single or a plurality of desired polynucleotide constructs using themethods described herein.

In some embodiments, the assembly procedure may include several paralleland/or sequential reaction steps in which a plurality of differentnucleic acids or oligonucleotides are synthesized or immobilized,amplified, and are combined in order to be assembled (e.g., by extensionor ligation as described herein) to generate a longer nucleic acidproduct to be used for further assembly, cloning, or other applications(see U.S. provisional application 61/235,677 and PCT applicationPCT/US09/55267 which are incorporate herein by reference in theirentirety). Amplification and assembly strategies provided herein can beused to generate very large libraries representative of many differentnucleic acid sequences of interest.

In some embodiments, methods of assembling libraries containing nucleicacids having predetermined sequence variations are provided herein.Assembly strategies provided herein can be used to generate very largelibraries representative of many different nucleic acid sequences ofinterest. In some embodiments, libraries of nucleic acid are librariesof sequence variants. Sequence variants may be variants of a singlenaturally-occurring protein encoding sequence. However, in someembodiments, sequence variants may be variants of a plurality ofdifferent protein-encoding sequences.

Accordingly, one aspect of the technology provided herein relates to thedesign of assembly strategies for preparing precise high-density nucleicacid libraries. Another aspect of the technology provided herein relatesto assembling precise high-density nucleic acid libraries. Aspects ofthe technology provided herein also provide precise high-density nucleicacid libraries. A high-density nucleic acid library may include morethat 100 different sequence variants (e.g., about 102 to 103; about 103to 104; about 104 to 105; about 105 to 106; about 106 to 107; about 107to 108; about 108 to 109; about 109 to 1010; about 1010 to 1011; about1011 to 1012; about 1012 to 1013; about 1013 to 1014; about 1014 to 1015or more different sequences) wherein a high percentage of the differentsequences are specified sequences as opposed to random sequences (e.g.,more than about 50%, more than about 60%, more than about 70%, more thanabout 75%, more than about 80%, more than about 85%, more than about90%, more than about 91%, more than about 92%, more than about 93%, morethan about 94%, more than about 95%, more than about 96%, more thanabout 97%, more than about 98%, more than about 99%, or more of thesequences are predetermined sequences of interest).

Some embodiments of the devices and methods provided herein useoligonucleotides that are immobilized on a support or substrate. As usedherein the term “support” and “substrate” are used interchangeably andrefers to a porous or non-porous solvent insoluble material on whichpolymers such as nucleic acids are synthesized or immobilized. As usedherein “porous” means that the material contains pores havingsubstantially uniform diameters (for example in the nm range). Porousmaterials include paper, synthetic filters and the like. In such porousmaterials, the reaction may take place within the pores. The support canhave any one of a number of shapes, such as pin, strip, plate, disk,rod, bends, cylindrical structure, particle, including bead,nanoparticle and the like. The support can have variable widths. Thesupport can be hydrophilic or capable of being rendered hydrophilic andincludes inorganic powders such as silica, magnesium sulfate, andalumina; natural polymeric materials, particularly cellulosic materialsand materials derived from cellulose, such as fiber containing papers,e.g., filter paper, chromatographic paper, etc.; synthetic or modifiednaturally occurring polymers, such as nitrocellulose, cellulose acetate,poly (vinyl chloride), polyacrylamide, cross linked dextran, agarose,polyacrylate, polyethylene, polypropylene, poly (4-methylbutene),polystyrene, polymethacrylate, poly(ethylene terephthalate), nylon,poly(vinyl butyrate), polyvinylidene difluoride (PVDF) membrane, glass,controlled pore glass, magnetic controlled pore glass, ceramics, metals,and the like etc.; either used by themselves or in conjunction withother materials. In some embodiments, oligonucleotides are synthesizedon an array format. For example, single-stranded oligonucleotides aresynthesized in situ on a common support wherein each oligonucleotide issynthesized on a separate or discrete feature (or spot) on thesubstrate. In preferred embodiments, single stranded oligonucleotidesare bound to the surface of the support or feature. As used herein theterm “array” refers to an arrangement of discrete features for storing,routing, amplifying and releasing oligonucleotides or complementaryoligonucleotides for further reactions. In a preferred embodiment, thesupport or array is addressable: the support includes two or morediscrete addressable features at a particular predetermined location (i.e., an “address”) on the support. Therefore, each oligonucleotidemolecule of the array is localized to a known and defined location onthe support. The sequence of each oligonucleotide can be determined fromits position on the support. Moreover, addressable supports or arraysenable the direct control of individual isolated volumes such asdroplets. The size of the defined feature can be chosen to allowformation of a microvolume droplet on the feature, each droplet beingkept separate from each other. As described herein, features aretypically, but need not be, separated by interfeature spaces to ensurethat droplets between two adjacent features do not merge. lnterfeatureswill typically not carry any oligonucleotide on their surface and willcorrespond to inert space. In some embodiments, features andinterfeatures may differ in their hydrophilicity or hydrophobicityproperties. In some embodiments, features and interfeatures may comprisea modifier as described herein.

Arrays may be constructed, custom ordered or purchased from a commercialvendor (e.g., Agilent, Affymetrix, Nimblegen). Oligonucleotides areattached, spotted, immobilized, surface-bound, supported or synthesizedon the discrete features of the surface or array Oligonucleotides may becovalently attached to the surface or deposited on the surface. Variousmethods of construction are well known in the art e.g. maskless arraysynthesizers, light directed methods utilizing masks, flow channelmethods, spotting methods etc.

In some embodiments, construction and/or selection oligonucleotides maybe synthesized on a solid support using maskless array synthesizer(MAS). Maskless array synthesizers are described, for example, in PCTapplication No. WO 99/42813 and in corresponding U.S. Pat. No.6,375,903. Other examples are known of maskless instruments which canfabricate a custom DNA microarray in which each of the features in thearray has a single-stranded DNA molecule of desired sequence.

Other methods for synthesizing construction and/or selectionoligonucleotides include, for example, light-directed methods utilizingmasks, flow channel methods, spotting methods, pin-based methods, andmethods utilizing multiple supports.

Light directed methods utilizing masks (e.g., VLSIPS™ methods) for thesynthesis of oligonucleotides is described, for example, in U.S. Pat.Nos. 5,143,854; 5,510,270 and 5,527,681. These methods involveactivating predefined regions of a solid support and then contacting thesupport with a preselected monomer solution. Selected regions can beactivated by irradiation with a light source through a mask much in themanner of photolithography techniques used in integrated circuitfabrication. Other regions of the support remain inactive becauseillumination is blocked by the mask and they remain chemicallyprotected. Thus, a light pattern defines which regions of the supportreact with a given monomer. By repeatedly activating different sets ofpredefined regions and contacting different monomer solutions with thesupport, a diverse array of polymers is produced on the support. Othersteps, such as washing unreacted monomer solution from the support, canbe optionally used. Other applicable methods include mechanicaltechniques such as those described in U.S. Pat. No. 5,384,261.

Additional methods applicable to synthesis of construction and/orselection oligonucleotides on a single support are described, forexample, in U.S. Pat. No. 5,384,261. For example, reagents may bedelivered to the support by either (1) flowing within a channel definedon predefined regions or (2) “spotting” on predefined regions. Otherapproaches, as well as combinations of spotting and flowing, may beemployed as well. In each instance, certain activated regions of thesupport are mechanically separated from other regions when the monomersolutions are delivered to the various reaction sites. Flow channelmethods involve, for example, microfluidic systems to control synthesisof oligonucleotides on a solid support. For example, diverse polymersequences may be synthesized at selected regions of a solid support byforming flow channels on a surface of the support through whichappropriate reagents flow or in which appropriate reagents are placed.Spotting methods for preparation of oligonucleotides on a solid supportinvolve delivering reactants in relatively small quantities by directlydepositing them in selected regions. In some steps, the entire supportsurface can be sprayed or otherwise coated with a solution, if it ismore efficient to do so. Precisely measured aliquots of monomersolutions may be deposited dropwise by a dispenser that moves fromregion to region.

Pin-based methods for synthesis of oligonucleotides on a solid supportare described, for example, in U.S. Pat. No. 5,288,514. Pin-basedmethods utilize a support having a plurality of pins or otherextensions. The pins are each inserted simultaneously into individualreagent containers in a tray. An array of 96 pins is commonly utilizedwith a 96-container tray, such as a 96-wells microtiter dish. Each trayis filled with a particular reagent for coupling in a particularchemical reaction on an individual pin. Accordingly, the trays willoften contain different reagents. Since the chemical reactions have beenoptimized such that each of the reactions can be performed under arelatively similar set of reaction conditions, it becomes possible toconduct multiple chemical coupling steps simultaneously.

In another embodiment, a plurality of oligonucleotides may besynthesized on multiple supports. One example is a bead based synthesismethod which is described, for example, in U.S. Pat. Nos. 5,770,358;5,639,603; and 5,541,061. For the synthesis of molecules such asoligonucleotides on beads, a large plurality of beads is suspended in asuitable carrier (such as water) in a container. The beads are providedwith optional spacer molecules having an active site to which iscomplexed, optionally, a protecting group. At each step of thesynthesis, the beads are divided for coupling into a plurality ofcontainers. After the nascent oligonucleotide chains are deprotected, adifferent monomer solution is added to each container, so that on allbeads in a given container, the same nucleotide addition reactionoccurs. The beads are then washed of excess reagents, pooled in a singlecontainer, mixed and re-distributed into another plurality of containersin preparation for the next round of synthesis. It should be noted thatby virtue of the large number of beads utilized at the outset, therewill similarly be a large number of beads randomly dispersed in thecontainer, each having a unique oligonucleotide sequence synthesized ona surface thereof after numerous rounds of randomized addition of bases.An individual bead may be tagged with a sequence which is unique to thedouble-stranded oligonucleotide thereon, to allow for identificationduring use.

In yet another embodiment, a plurality of oligonucleotides may beattached or synthesized on nanoparticles. Nanoparticles includes but arenot limited to metal (e.g., gold, silver, copper and platinum),semiconductor (e.g., CdSe, CdS, and CdS coated with ZnS) and magnetic(e.g., ferromagnetite) colloidal materials. Methods to attacholigonucleotides to the nanoparticles are known in the art. In anotherembodiment, nanoparticles are attached to the substrate. Nanoparticleswith or without immobilized oligonucleotides can be attached tosubstrates as described in, e.g., Grabar et al., Analyt. Chem., 67,73-743 (1995); Bethell et al., J. Electroanal. Chem., 409, 137 (1996);Bar et al., Langmuir, 12, 1172 (1996); Colvin et al., J. Am. Chem. Soc.,114, 5221 (1992). Naked nanoparticles may be first attached to thesubstrate and oligonucleotides can be attached to the immobilizednanoparticles.

Pre-synthesized oligonucleotide and/or polynucleotide sequences may beattached to a support or synthesized in situ using light-directedmethods, flow channel and spotting methods, inkjet methods, pin-basedmethods and bead-based methods set forth in the following references:McGall et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93:13555; SyntheticDNA Arrays In Genetic Engineering, Vol. 20:111, Plenum Press (1998);Duggan et al. (1999) Nat. Genet. S21:10; Microarrays: Making Them andUsing Them In Microarray Bioinformatics, Cambridge University Press,2003; U.S. Patent Application Publication Nos. 2003/0068633 and2002/0081582; U.S. Pat. Nos. 6,833,450, 6,830,890, 6,824,866, 6,800,439,6,375,903 and 5,700,637; and PCT Publication Nos. WO 04/031399, WO04/031351, WO 04/029586, WO 03/100012, WO 03/066212, WO 03/065038, WO03/064699, WO 03/064027, WO 03/064026, WO 03/046223, WO 03/040410 and WO02/24597; the disclosures of which are incorporated herein by referencein their entirety for all purposes. In some embodiments, pre-synthesizedoligonucleotides are attached to a support or are synthesized using aspotting methodology wherein monomers solutions are deposited dropwiseby a dispenser that moves from region to region (e.g. ink jet). In someembodiments, oligonucleotides are spotted on a support using, forexample, a mechanical wave actuated dispenser.

The manipulation of fluids to form fluid streams of desiredconfiguration, such as discontinuous fluid streams, particles,dispersions, etc., for purposes of fluid delivery, product manufacture,analysis, and the like, is a relatively well-studied art. See forexample, WO/2004/002627 which is incorporated herein in its entirety. Insome aspects of the invention, microfluidic devices are used to form andmanipulate droplets in a co-planar fashion to allow oligonucleotidesynthesis. For example, oligonucleotides may be synthesized using aphophoramidite method. The phosphoramidite method, employing nucleotidesmodified with various protecting groups, is one of the most commonlyused methods for the de novo synthesis of oligonucleotides. Detailedprocedures for the phosphoramidite and hydrogen phosphonate methods ofoligonucleotide synthesis are described in the following references thatare incorporated by reference: U.S. Pat. Nos. 4,500,707; 4,725,677; and5,047,524. See also for example, methods outlined in Oligonucleotide andAnalogs: A practical approach, F. Eckstein, Ed. IRL Press OxfordUniversity and Oligonucleotide synthesis: A practical approach, Gait,Ed. IRL Oxford Press. Synthesis can be performed either through thecoupling of the 5′ position of the first monomer to the 3′ position ofthe second monomer (3′-5′ synthesis) or vive versa (5′-3′ synthesis).Briefly, synthesis of oligonucleotides requires the specific formationof a 3′-5′ or 5′3′ phosphodiester linkage. In order to form thesespecific linkages, the nucleophilic centers not involved in the linkagemust be chemically protected through the use of protecting group. By“protecting group” as used herein is meant a species which prevents asegment of a molecule (e.g. nucleotide) from undergoing a specificchemical reaction, but which is removable from the molecule followingcompletion of that reaction. For example, the 5′ hydroxyl group may beprotected by dimethoxitrityl (DMT). During the deblocking reaction, theDMT is removed with an acid, such as thrichloroacetic acid (TeA) ordichloroacetic acid, resulting in a free hydroxyl group. After washing,a phosphoramidite nucleotide is activated by tetrazole,ethylthiotetrazole, dicyanoimidazole, or benzimidazolium triflate, forexample, which remove the iPr2N group on the phosphate group. Thedeprotected 5′ hydroxyl of the first base reacts with the phosphate ofthe second base and a 5′-3′ linkage is formed (coupling step). Unboundbases are washed out and 5′ hydroxyl group that did not react during thecoupling reaction are blocked by adding a capping group, whichpermanently binds to the free 5′ hydroxyl groups to prevent any furtherchemical transformation of that group (capping step). The oxidation stepmay be performed before or after the capping step. During oxidation, thephosphite linkage is stabilized to form a much more stable phosphatelinkage. The deblocking/coupling/capping/oxidation cycle may be repeatedthe requisite number of time to achieve the desired lengthpolynucleotide. In some embodiments, coupling can be synchronized on thearray or solid support.

In some embodiments, the oligonucleotides synthesis is synthesized usinga device that generates emulsion droplets comprising aqueous dropletswithin immiscible oil. The droplets may comprise an aqueous phase, animmiscible oil phase, and a surfactant and/or other stabilizingmolecules to maintain the integrity of the droplet. In some embodiment,mechanical energy is applied, allowing dispersion of a compound into anoil phase to form droplets, each of which contains a single sort ofcompound. Preferably, the compound is a nucleotide monomer (i.e. A, TorU, G C). The compounds can be deposited into the oil phase in the formof droplets generated using inkjet printing technology or piezoelectricdrop-on-demand (DOD) inkjet printing technology. Each droplet maycomprise a different nucleotide monomer (i.e. A, T or U, G C) in thesame aqueous solution. In preferred embodiments, the droplets areuniform in size and contain one nucleotide at a fixed concentration. Thedroplets range in size from 0.5 microns to 500 micron in diameter, whichcorrespond to a volume of about 1 picoliter to about 1 nanoliter. Yet inother embodiments, the droplet may comprise a 2-mer, a 3-mer, a 4-mer, a6-mer or a 7-mer oligonucleotide. In some embodiments, the droplets aredeposited onto a substrate such as a microsubstrate, a microarray or amicrochip. The terms microsubstrate, microarray and microchip are usedinterchangeably herein. The droplets may be deposited using amicrofluidic nozzle. In some embodiments, the substrate may be subjectedto wash, deblocking solution, coupling, capping and oxidation reactionsto elongate the oligonucleotide.

In some embodiments, the droplets carrying the nucleotides can me movedusing electrowetting technologies. Electrowetting involves modifying thesurface tension of liquids on a solid surface using a voltage.Application of an electric field (e.g. alternating or direct), thecontact angle between the fluid and surfaces can be modified. Forexample, by applying a voltage, the wetting properties of a hydrophobicsurface can become increasingly hydrophilic and therefore wettable.Electrowetting principle is based on manipulating droplets on a surfacecomprising an array of electrodes and using voltage to change theinterfacial tension. In some embodiments, the array of electrode is notin direct contact with the fluid. In some embodiments, the array ofelectrode is configured such as the support has a hydrophilic side and ahydrophobic side. The droplets subjected to the voltage will movetowards the hydrophilic side. In some embodiments, the array or patternof electrodes is a high density pattern. One should appreciate that tobe used in conjunction with the phophoramidite chemistry, the array ofelectrodes should be able to move droplets volumes ranging from 1 pL(and less) to 10 pL. Accordingly, aspects of the invention relate tohigh voltage complementary semi-conductor microfluidic controller. Insome embodiments, the high voltage complementary semi-conductor device(HV-CMOS) has an integrated circuit with high density electrode patternand high voltage electronics. In some embodiments, the voltage appliedis between 15V and 30V.

Methods and devices provided herein involve amplification and/or smallassembly reaction volumes such as microvolumes, nanovolumes, picovolumesor sub-picovolumes. Accordingly, aspects of the invention relate tomethods and devices for amplification and/or assembly of polynucleotidesequences in small volume droplets on separate and addressable featuresof a support. For example, a plurality of oligonucleotides complementaryto surface-bound single stranded oligonucleotides is synthesized in apredefined reaction microvolume of solution by template-dependantsynthesis. In some embodiments, predefined reaction microvolumes ofbetween about 0.5 pL and about 100 nL may be used. However, smaller orlarger volumes may be used. In some embodiments, a mechanical waveactuated dispenser may be used for transferring volumes of less than 100nL, less than 10 nL, less than 5 nL, less than 100 pL, less than 10 pL,or about 0.5 pL or less. In some embodiments, the mechanical waveactuated dispenser can be a piezoelectric inkjet device or an acousticliquid handler. In a preferred embodiment, a piezoelectric inkjet deviceis used and can deliver picoliter solutions in a very precise manner ona support.

Aspects of the invention relate to the manipulation of sub-microvolumeson a substrate and to the control of the movement of micro-volumes on asubstrate. It is a well-known phenomenon that the surfaces of mostnormally solid substrates, when contacted with a solution, have acharacteristic degree of non-wettability. That is, aqueous solutions donot spread on the solid surface but contract to form droplets.Accordingly, preferable supports have surface properties, primarilysurface tension and wettability properties that allow droplet formationwhen small volumes are dispensed onto the addressable feature. In someembodiments, the microvolume is bounded completely or almost completelyby the free surface forming a droplet or microdrop. One skilled in theart will understand that the shape of the droplet will be governed andmaintained by the contact angle of the liquid/solid interaction, surfacetension of the liquid as well as by surface energy. Adhesive forcesbetween a liquid and solid will cause a liquid drop to spread across thesurface whereas cohesive forces within the liquid will cause the drop toball up and avoid contact with the surface. For liquid, the surfaceenergy density is identical to the surface tension. Surface tension isthat property of matter, due to molecular forces, which exists in thesurface film of all liquids and tends to bring the contained volume intoa form having the least possible superficial area. In some embodiments,the support's surface is partitioned into discrete regions where thesurface contact angles of at least a plurality of the discrete regiondiffer for the fluid of interest. As used herein the term “contactangle” refers to a quantitative measure of the wetting of a solid by aliquid. A contact angle is defined as the angle formed by a liquid atthe three phase boundary where vapor (gas, e.g., atmosphere), liquid andsolid intersect. For example, in the case of a micro-volume dropletdispensed on a horizontal flat surface, the shape of the micro-volumedroplet will be determined by the Young equilibrium equation:

0=γ_(SV)−γ_(SL)−γ cos θ_(C)

wherein γ_(SV) is the solid-vapor interfacial energy; γ_(SL) is thesolid-liquid interfacial energy and γ is the liquid-vapor energy (i.e.surface tension) and ⊖_(C) is the equilibrium contact angle.

It will be understood that for contact angle values ⊖C less than 90°,the liquid will spread onto the solid surface. For example, veryhydrophilic surfaces have a contact angle of 0° to about 30°. In thecase of aqueous solutions and highly hydrophilic support, the contactangle ⊖C will be close to 0°, and the aqueous solution or droplet willcompletely spread out on the solid surface (i.e., complete wetting ofthe surface). On the contrary, for contact angle values ⊖C equal to orgreater than 90°, the liquid will rest on the surface and form a dropleton the solid surface. The shape of the droplet is determined by thevalue of the contact angle. In the case of aqueous solutions and highlyhydrophobic surfaces, liquid will bead up. In some embodiments, thesupport is chosen to have a surface energy and surface contact anglethat does not allow the droplets to spread beyond the perimeter of thefeature. Furthermore, on an ideal surface each droplet will return toits original shape if it is disturbed, for example after addition of amiscible or non-miscible solution. In some embodiments, the surface ispartitioned into regions where the surface contact angles of the regionsdiffer for the liquid of interest. In some embodiments, theses regionscorrespond to the discrete features of the substrate. In a preferredembodiment, the surface is partitioned into regions by modifiers.Modifiers may be added to specific locations of the substrate's surface.In some cases, the surface will be partitioned into regions comprisingmodifiers and unmodified surface areas. In some embodiments, thenon-modifier regions correspond to the unmodified substrate. Yet, inother embodiments, the non-modifiers regions correspond to a surface ofany arbitrary modification or any modifier that is different than themodifier a region that corresponds to a feature of a support. In someembodiments, the modifiers are oligomers. For example, the modifierscorrespond to nucleic acids and are modifying a set of discrete featuresof the substrate. As shown in FIG. 1A-C, the surface comprises a patternof modifiers and non-modifier regions having different surface contactangles. Exemplary patterning (or partitioning) of the surface is shownin FIG. 1A-C. Modifiers can have circular, square, trapezoid, or anygeometrical shape or any combination thereof. In some embodiments,modifiers are arranged in a grid-like pattern or in any other differentconfigurations. Pattern examples are shown in FIG. 1C. However, thepattern needs not to be restricted to any design. For example, themodifiers may be arranged in a randomly formed pattern. Patterning maybe formed by any process known in the art. For example, arrangedpatterning or random patterning may be formed by processes such as blockco-polymer surface self assembly. In other embodiments, the substratesurface is partitioned into regions by at least two different modifiersregions as discussed herein. In some embodiments, the surface contactangle of the modifiers (⊖M) is different than the surface contact angleof the non-modifier region (⊖NM). For example, the surface contact angleof the modifiers may be greater than the surface contact angle of thesurface or the non-modifier regions (⊖M>⊖NM). Alternatively, the surfacecontact angle of the non-modifier regions is greater than the surfacecontact angle of the modifiers (⊖M<⊖NM). In the context of aqueoussolutions, the modifiers surfaces may be more hydrophilic than thesurface of the non-modifiers regions (i.e. surface contact angle of themodifiers is smaller than the surface contact angle of the surface ornon-modifier regions). Alternatively, the modifiers surfaces may be morehydrophobic than the surface of the non-modifiers surface regions (i.e.,surface contact angle of the modifiers is smaller than the surfacecontact angle of the surface or non-modifier regions). In an exemplaryembodiment, modifiers are oligonucleotides and the surface of themodifier regions is more hydrophilic than the surface of thenon-modifier regions. In other embodiments, the totality or asubstantial part of the support or surface is covered with at least twodifferent modifiers, such as for example a first and a second modifier(for example, modifiers (3) and (4) as shown in FIG. 1A-ii). The atleast two different modifiers may be patterned as described above. Forexample, the first and second modifiers can cover the surface in analternative pattern as shown in FIG. 1 A-C. One should appreciate thatthe support surface may be covered with a plurality of modifiers thatare disposed on the surface to form a hydrophilic gradient. In someembodiments, each modifier has a different contact angle than theadjacent modifier. In some embodiments, the surface is partitioned witha plurality of different modifiers, the plurality of first modifiersbeing more hydrophilic than the at least one second modifier, and theplurality of first modifiers having each a slightly different contactangle than the next first modifier. For example, the contact angle ofeach of the plurality of first modifier may differ by at least about 1°,2°, 3°, 4°, 5°, 6°, 7°, 8°, 9°, 10° 11°, 12°, 13,° 14°, 15°, 16°, 17°,18°, 19°, 20°, 25°, 30° or more from that of the next first modifier.The plurality of first modifiers therefore forms a hydrophilic gradientand a predetermined path along which a droplet can be moved bysurface-tension manipulation.

According to some aspects of the invention, the difference in surfacecontact angles between two different modifiers or a modifier and thenon-modifier surface creates a virtual “wall”. As used herein the termsmaller contact angle (SCA) refers to the surface or modifier havingsmaller contact angle and the term higher contact angle (HCA) refers tothe surface or modifier having higher contact angle. In the context ofaqueous solutions, SCA are more hydrophilic than HCA. In someembodiments, HCA values are at least 20°, at least 30°, at least 35°higher than SCA. Accordingly, liquid volumes can be formed and isolatedon surfaces comprising regions of SCA and regions of HCA. For example,if the surface contact angle of the modifier is greater than thenon-modifier surface contact angle, liquid volumes will form a dropletbetween two modifiers regions (see FIG. 2A). One would appreciate thatdepending on liquid volume deposited onto the surface and the differenceof contact values between modifiers, the droplet can occupy a singleregion of small contact angle (as shown by droplet 11 in FIG. 2) ormultiple regions (as shown by droplet 12 in FIG. 2A). For example, theliquid volume may occupy 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more SCAregions. Accordingly, the liquid can occupy a footprint corresponding toone or more SCA. The footprint may then encompass one or more HCA. Insome embodiments, to ensure that two droplets or small isolated volumeswill not merge, liquid volumes are placed sufficiently apart from eachothers. For example, the spacing between two isolated volumes maycomprise at least one, at least two, at least three, at least four, atleast five, at least six, at least seven, at least eight, at least nine,at least ten HCA regions or modifiers regions. Placing the liquidvolumes sufficiently apart also allows for keeping liquid volumesisolated during fluctuation of temperature such as during thermocycling.Because surface tension usually decreases with the increase oftemperature, droplets may spread or move on the surface when thetemperature of the support or of the liquid volume is raised. It will beappreciate that if the liquid volumes are kept sufficiently apart,liquid volumes will remain isolated and will not merge with adjacentliquid volumes during fluctuation of the temperature.

In one aspect of the invention, methods and devices are provided forprocessing independently one or more plurality of oligonucleotides in atemperature dependent manner at addressable features in isolated liquidvolumes. In some embodiments, the method is conducted in a manner toprovide a set of predefined single-stranded oligonucleotide sequences orcomplementary oligonucleotide sequences for further specified reactionsor processing. One should appreciate that each features beingindependently addressable, each reaction can be processed independentlywithin a predefined isolated liquid volume or droplet on a discretefeature (e.g. virtual chamber). In some embodiments, the arrays arestored dry for subsequent reactions. In a preferred embodiment, supportimmobilized oligonucleotides can be hydrated independently with anaqueous solution. Aqueous solution includes, but is not limited to,water, buffer, primers, master mix, release chemicals, enzymes, or anycombination thereof. Aqueous solution can be spotted or jetted ontospecific surface location(s) corresponding to the discrete feature(s).Subsequently, miscible as well as non-miscible solution or aqueous gelcan be deposited in the same fashion. Alternatively, a mechanical waveactuated dispenser can be used for transferring small volumes of fluids(e.g., picoliter or sub-picoliter). A mechanical wave actuated dispensercan be a piezoelectric inkjet device or an acoustic liquid handler. Apiezoelectric inkjet device can eject fluids by actuating apiezoelectric actuation mechanism, which forces fluid droplets to beejected. Piezoelectrics in general have good operating bandwidth and cangenerate large forces in a compact size. Some of the commerciallyavailable piezoelectric inkjet microarraying instruments include thosefrom Perkin Elmer (Wellesley, Mass.), GeSim (Germany) and MicroFab(Plano, Tex.). Typical piezoelectric dispensers can create droplets inthe picoliter range and with coefficient of variations of 3-7%.Inkjetting technologies and devices for ejecting a plurality of fluiddroplets toward discrete features on a substrate surface for depositionthereon have been described in a number of patents such as U.S. Pat.Nos. 6,511,849; 6,514,704; 6,042,211; 5,658,802, the disclosure of eachof which is incorporated herein by reference.

In one embodiment, the fluid or solution deposition is performed usingan acoustic liquid handler or ejector. Acoustic devices are non-contactdispensing devices able to dispense small volume of fluid (e.g.picoliter to microliter), see for example Echo 550 from Labcyte (CA),HTS-01 from EDC Biosystems. Acoustic technologies and devices foracoustically ejecting a plurality of fluid droplets toward discretesites on a substrate surface for deposition thereon have been describedin a number of patents such as U.S. Pat. Nos. 6,416,164; 6,596,239;6,802,593; 6,932,097; 7,090,333 and US Patent Application 2002-0037579,the disclosure of each of which is incorporated herein by reference. Theacoustic device includes an acoustic radiation generator or transducerthat may be used to eject fluid droplets from a reservoir (e.g.microplate wells) through a coupling medium. The pressure of the focusedacoustic waves at the fluid surface creates an upwelling, therebycausing the liquid to urge upwards so as to eject a droplet, for examplefrom a well of a source plate, to a receiving plate positioned above thefluid reservoir. The volume of the droplet ejected can be determined byselecting the appropriate sound wave frequency.

In some embodiments, the source plate comprising water, buffer, primers,master mix, release chemicals, enzymes, or any combination thereof andthe destination plates comprising the oligonucleotides orpolynucleotides are matched up to allow proper delivery or spotting ofthe reagent to the proper site. The mechanical wave actuated dispensermay be coupled with a microscope and/or a camera to provide positionalselection of deposited spots. A camera may be placed on both sides ofthe destination plate or substrate. A camera may be used to register tothe positioning on the array especially if the DNA is coupled with afluorescent label. As shown in FIG. 7A and described below components ofthe device include: 101: Source well plate; containing the reagents forreactions, this element can travel in at least 2 degree-of-freedom(>2DOF); 102: Solid support, with solid attached molecules, this elementcan travel in at least 2 degree-of-freedom (>2DOF); 103: Transducer, tocreate a mechanical wave which causes droplets to form and travel, thiselement can travel in at least 2 degree-of-freedom (>2DOF); 104:Coupling fluid, to allow the mechanical wave to couple to the well plate(101); 105: Reagents inside a well on the source well plate (101); 171:Camera (such as an electronic camera) used to provide physicalregistration (positioning) and 172: Surface mark fixed on the solidsupport (102) to provide a reference position on the solid support. FIG.7B shows another example where an inkjet device 180 (e.g.,piezoelectric) can be used to dispense droplets.

As illustrated in FIGS. 7A and 7B, a mechanical wave actuated dispenser(103, FIG. 7A, 180, FIG. 7) can be used to create traveling droplets(106) from a reagent source (101). The created traveling droplets (106)can be deposited onto a receiving surface, in this case a solid support(102). The position of the deposited droplets (111) on the solid support(102) can be controlled by the relative position of 101 and 102.Furthermore, there can be an existing pattern of molecules (110) on thesolid support (102). The traveling droplets (106) can be aligned to theexisting pattern on the surface. One should appreciate that thealignment and the dispensing are crucial steps in some embodiments.Multiple reagents can be dispensed to the sites on the surface in asequential process. The solid support (102) is also known as thedestination surface. This surface may have molecules that are previouslydeposited on the surface. These molecules can represent a complexpattern on the surface of (102). These molecules may be covalentlybonded, hydrogen bonded, or not bonded (just deposited in solution ordry form) to the surface of (102). In a preferred embodiment, thedroplets (106) created by the mechanical wave actuated dispenser (103,180) are aimed and deposited at desired positions on the surface of 102.Adjacent surface droplets (111) can be combined by the creation of“merger” droplets (112) by positioning the merger droplets (112) inbetween or around the surface droplets (111). In this embodiment, thealignment of the droplets (106) to the solid support (102) and themolecules attached to the solid support (110) is crucial. A system canbe devised to align the droplet to the patterns (molecules) on 102 byusing a variety of sensing methods. In some embodiments, the position ofthe dispensed droplet in relation to the existing pattern on 102 isknown or determinable by the user. The detection method and devices canbe based on acoustics, electrical conductive, electric capacitive, oroptical sensors.

In FIG. 7A, the alignment is illustrated using an optical setup. A setof test droplets (170) can be dispensed to several locations on thesolid support (102) dispensed for the purpose of alignment of thedroplet to the solid attached molecules (111). The relative positionbetween the test droplets (170) and fixed registration marks (172: asurface mark fixed on the solid support (102) to provide a referenceposition on the solid support) on the surface of 102 can provideinformation on the alignment between the source plate (101) and thedestination surface (102). A computer system can be used to calculate aset of correction (offset) parameters, which will be used to correct thealignment by adjusting the positioning motors controlling the positionof 101, 102, and 103.

One should appreciate that when manipulating small liquid volumes suchas picoliters and nanoliters, the smaller the droplet, the faster itwill evaporate. Therefore, aspects of the invention relate to methodsand devices to limit, retard or prevent water evaporation. In someembodiments, the discrete features or a subset of discrete features canbe coated with a substance capable of trapping or capturing water. Inother embodiments, the water-trapping material can be spin-coated ontothe support. Different materials or substances can be used to trap waterat specific locations. For example, the water trapping substance may bean aqueous matrix, a gel, a colloid or any suitable polymer. In someembodiments, the material is chosen to have a melting point that allowsthe material to remain solid or semi-solid (e.g. gel) at the reactiontemperatures such as denaturing temperatures or thermocyclingtemperatures. Water trapping materials include but are not limited tocolloidal silica, peptide gel, agarose, solgel and polydimethylsiloxane.In an exemplary embodiment, Snowtex® colloidal silica (Nissan Chemical)may be used. Snowtex colloidal silica is composed of mono-dispersed,negatively charged, amorphous silica particles in water. Snowtexcolloidal silica can be applied as dry gel or as an hydrated gel ontothe surface. In a preferred embodiment, the water trapping substance isspotted at discrete features comprising surface-bound oligonucleotides.Alternatively, oligonucleotides can be synthesized on the particles ornanoparticles (e.g. colloidal particles, Snowtex colloidal silica) andthe particles or nanoparticles can be dispensed to the discrete featuresof the surface. In some embodiments, the water trapping substance isspotted on the discrete features of the support using a mechanicaldevice, an inkjet device or an acoustic liquid handler.

One should appreciate, that evaporation can also be limited by forming aphysical barrier between the surface of the droplet and the atmosphere.For example, a non-miscible solution can be overlaid to protect thedroplet from evaporation. In some embodiments, a small volume of thenon-miscible solution is dispensed directly and selectively at discretelocation of the substrate such as features comprising a droplet. In someother embodiments, the non-miscible solution is dispensed onto a subsetof features comprising a droplet. In other embodiments, the non-misciblesolution is applied uniformly over the surface of the array forming anon-miscible bilayer in which the droplets are trapped. The non-misciblebilayer can then be evaporated to form a thin film over the surface orover a substantial part of the surface of the droplet. The non-misciblesolution includes, but is not limited to, mineral oil, vegetable oil,silicone oil, paraffin oil, natural or synthetic wax, organic solventthat is immiscible in water or any combination thereof. One skilled inthe art will appreciate that depending on the composition of the oils,some oils may partially or totally solidify at or below roomtemperature. In some embodiments, the non-miscible solution may be anatural or synthetic wax such as paraffin hydrocarbon. Paraffin is analkane hydrocarbon with the general formula CnH2n+2. Depending on thelength of the molecule, paraffin may appear as a gas, a liquid or asolid at room temperature. Paraffin wax refers to the solids with20<n<40 and has a typical melting point between about 47° C. to 64° C.Accordingly, in some embodiments, the support may be stored capped witha wax. Prior to use, heat may be applied to the support to allow the waxto turn into a liquid wax phase coating the support.

In some aspects of the invention, in subsequent steps, a solvent or anaqueous solution may be added to the droplet having a non-misciblesolution at its surface. Aqueous solution may be added, for example, toinitiate a reaction, to adjust a volume, to adjust a pH, to increase ordecrease a solute concentration, etc. . . . One would appreciate thatthe aqueous solution can penetrate the non-miscible layer usingdifferent mechanisms. For example, if using an inkjet head device, theaqueous solution is ejected and the physical momentum of the ejecteddroplet will enable the aqueous solution to cross the non-misciblelayer. Other mechanisms may employ additional forces, such as forexample magnetic and/or electrostatic forces and/or optical forces. Theoptical and magnetic forces can be created simultaneously orindependently of one another. Furthermore, the mechanism can utilizecoupled magneto-optical tweezers. In some embodiments, the aqueoussolution to be dispensed contains magnetic nanoparticles and a magneticforce can be used to help penetration of the non-miscible layer.Alternatively, the aqueous solution carries an electrostatic charge andan external applied electric field can be used to achieve penetration ofthe non-miscible layer.

Yet, in another aspect of the invention, the size of the droplet iscontinuously or frequently monitored. One should appreciate that thesize of the droplet is determined by the volume and by the surfacetension of the solution. Accordingly, loss of volume can be detected bya decrease of the droplet footprint or radius of the droplet footprint.For example, using an optical monitoring system, through a microscopelens and camera system, the size or footprint of the droplet can bedetermined and the volume of the droplet can be calculated. In someembodiments, the volume of the droplet or the radius of the droplet ismonitored every second or every millisecond. One would appreciate thatthe magnitude of the evaporation rate of the solvent (e.g. water) fromthe droplet of interest depends in part on the temperature and thusincreases with increasing temperatures. For example, duringamplification by thermocycling or during denaturation of thedouble-stranded complexes, increase of temperature will result in therapid evaporation of the droplet. Therefore, the volume of the dropletcan be monitored more frequently and the droplet volume can be adjustedby re-hydration more frequently. In the event of volume fluctuation suchas loss of volume, sub-pico to nano volumes of solvent (e.g. water) canbe dispensed onto the droplet or to the discrete feature comprising thedroplet. Solvent or water volumes of about 0.5 pL, of about 1 pL, ofabout 10 pL, of about 100 pL, of about 1 nL, of about 10 nL, of about100 nL can be dispensed this way. Solvent or water volumes may bedelivered by any conventional delivery means as long that the volumesare controlled and accurate. In a preferred embodiment, water isdispensed using an inkjet device. For example, a typical inkjet printeris capable of producing droplets volumes ranging from about 1.5 pL toabout 10 pL, while other commercial ultrasonic dispensing techniques canproduce droplets volumes of about 0.6 pL. In some embodiments, water isadded in a rapid series of droplets. In some embodiments, water isdispensed when registering a loss of volume of more than 10%, of morethan 25%, of more than 35%, of more than 50%.

In another embodiment, the evaporation rate can be limited by adding acompound having a high boiling point component to the droplet(s) ofinterest, provided that the presence of the compound does not inhibitthe enzymatic reactions performed on the substrate. The boiling point ofa liquid is the temperature at which the liquid and vapor phases are inequilibrium with each other at a specified pressure. When heat isapplied to a solution, the temperature of the solution rises until thevapor pressure of the liquid equals the pressure of the surroundinggases. At this point, vaporization or evaporation occurs at the surfaceof the solution. By adding a high boiling point liquid to the droplet ofinterest, evaporation of the water content of a droplet may besubstantially reduced (see U.S. Pat. No. 6,177,558). In some embodiment,the high boiling point solution is a solvent. In some embodiments, thehigh boiling point liquid has a boiling point of at least 100° C., atleast 150° C., at least 200° C. In some embodiments, glycerol is addedto the solution, increasing the boiling point. Accordingly, the solutioncontaining the high boiling point liquid will evaporate at a much slowerrate at room temperature or at reaction conditions such asthermocycling, extension, ligation and denaturation.

In other embodiments, evaporation rate is limited by raising the vaporrate or humidity surrounding the droplet. This can be performed, forexample, by placing “sacrificial” droplets around or in close proximityto the droplet of interest (e.g. around or in close proximity of adroplet comprising the oligonucleotides) (see for example, Berthier E.et al., Lab Chip, 2008, 8(6):852-859).

Some aspects of the invention relate to devices to control the humidityand/or the evaporation rate. In some embodiments, the surface or solidsupport is enclosed in a closed container to limit the evaporation. FIG.23A and FIG. 23B illustrate non-limiting embodiments of a microvolumesealed plates. Referring to FIG. 23 A, a substrate or support (1502) isprovided, the substrate having defined features (1508) such that volumesof reactions (1503, 1504, 1505, 1506, 1507) can be deposited in suchfeatures via, for example, an inkjet and inkjet-like liquid dispensingtechnology. A lid (1501) is used to seal such reaction volumes by eitherapplied pressure or using a lid (1501) with a pressure-sensitiveadhesive on the contacting side to the substrate (1502). In someembodiments, the density of these features can be at least 10 featuresper cm², at least 100 features per cm², at least 1,000 features per cm²,at least 10,000 features per cm², at least 100,000 features per cm², atleast 1,000,000 features per cm². The features (1508) can have diameter(width and length) dimensions from less than about 1 cm, less than about1 mm, less than about 1 μm. The depth of the features can havedimensions from less than about 1 cm, less than about 1 mm, less thanabout 1 μm. The width, length, and depths of the features can differfrom feature to feature. The features geometry can be complex, includinglines, spirals, bends (at all possible angles from 0.01 degrees to179.99 degrees) or any combination of such complex geometries.

In another embodiment, the substrate is flat (1602) with reactionvolumes (e.g. droplets 1604) set up on a surface of the substrate. Thelid (1601) is designed to have features that form containers or vesselsfor reaction volumes (1604). The reaction volumes (1604) can be createdon the substrate (1602) using inkjet and inkjet-like liquid manipulationtechnologies. The lid (1601) can be sealed against the substrate (1602)by either applied pressure or using a lid (1601) or substrate (1602)with a pressure-sensitive adhesive on the contacting side to thesubstrate (1602).

Aspects of the invention relate to feedback controlled humidity devices,systems and methods. In some embodiments, the device comprise aconfinement chamber structure. Referring to FIG. 24A, a volume (1709) ofa mixture of different gases, such oxygen, nitrogen, argon, helium,water vapor, solvent vapor, and any other desirable gases, can bemaintained inside a confinement chamber structure (1701) consisting ofwalls (1706). In some embodiments, openings (1705) on the wall (1706)allow introduction and removal of different components of the gases toachieve the desired composition of the gas mixture in the volume (1709).Additional openings (1704) can be used to serve as measurement orsampling ports to examine the condition or composition of the gas in thevolume. A substrate (1702) carrying small reaction volumes (e.g.droplets 1703) deposited by, for example, an inkjet or inkjet-likeliquid dispensing technology can be placed inside the chamber's volume(1709).

The chamber's volume can be further confined by a lid (1707). In someembodiments, the lid is temperature controlled. The lid can be made of amaterial that is optically transparent, such as glass. The heating ofthe lid can be accomplished via an electrically conductive layer ofIndium in Oxide (ITO), and heated via Ohmic heating. Other heating orcooling methods are also possible, for example, via forced fluid flow.The chamber's volume can be further confined by a bottom (1708). In someembodiment, the bottom is temperature controlled. In some embodiments,the volume (1709) is modulated to contain an environment that has theexact molar ratio of different gas mixtures. In a preferred embodiment,the molar ratio of water vapor and carrier gas (air, helium, argon,nitrogen, or any other desirable gas, including solvent vapors) can becontrolled, together with the temperature of the volume (1709), to allowan equilibrium between water or solvent evaporation and condensation onthe surface of the substrate (1702). This equilibrium allows thereaction volumes (1703) on the substrate (1702) to be maintained at thedesirable steady volume over an appropriate period of time. Theappropriate period of time can be in the range of seconds, minutes,hours or days. One skilled in the art would appreciate that the dropletsvolumes can be maintained, decreased or increased by controllingevaporation and/or condensation. For example evaporation of the reactionvolumes (1703) on the substrate is induced to, for example, achieveand/or control sample concentration and/or decrease the reactionvolumes. Yet in other embodiments, condensation of the reaction volumes(1703) on the substrate is induced to, for example, achieve and/orcontrol sample dilution and/or increase reaction volumes. One wouldappreciate that it is important to control condensation when increasingreaction volumes. In some embodiments, condensation can be controlled byperiodic humidity compensation. For example, by increasing thetemperature on the substrate and/or lowering the humidity in thechamber, evaporation can be induced over a short period of time (in therange of ms, s or min). The evaporation of small satellite droplets(e.g. off target droplets) will take place before evaporation of largerdroplets (e.g. reaction volumes). Since the evaporation rate (by volume)is proportional to droplets' surface areas, and smaller droplets havinghigher surface-to-volume ratio evaporate first. In other embodiments,condensation may be controlled by controlling substrate's surfaceproperties such as hydrophilicity/hydrophobicity. One skilled in the artwill appreciate that condensation or droplet growth is characterized bynucleation of the droplet at nucleation sites. The rate of nucleation isa function of the surface tension and the wetting angle. Accordingly,surfaces promoting nucleation have a wetting-contact angles greater thanzero. In some embodiments, condensation can be controlled by designingoff-target areas on the surface (such as interfeatures) having surfaceproperties impairing nucleation. For example, the substrate's surfacecan be treated so that off target areas are more hydrophobic than areaswhere droplet growth is desired. In other embodiments, the off-targetareas surfaces are designed to be smooth so that no nucleation isreduced.

In some aspects of the invention, the reaction volumes are controlledvia a feedback control. In some embodiments, one or more monitoringisolated volumes (e.g. monitoring droplets) are used to monitor aplurality of isolated reaction volumes (e.g. droplets comprisingpredefined oligonucleotide sequences) on a support. In some embodiments,a first support is provided which comprises a plurality of reactiondroplets and a second support is provided comprising at least onemonitoring droplet. Preferably, the at least one monitoring droplet hasan identical surface-to-volume ratio than at least one of the reactiondroplet of interest and an identical solvent composition. Accordingly,modification of the reaction volume of the monitoring droplet isindicative of the modification of volume of the at least one droplet ofinterest. In some embodiments, the reaction droplets and monitoringdroplets are placed on the same support.

In some embodiments, the molar ratio of the mixture of gases is measuredusing a cold mirror setup. As illustrated in FIG. 24C, an opticallyreflective surface (1770) can be placed on the bottom surface (1708),next to the substrate (1702). The mirror can be of a similar materialand similar thickness to the substrate (1702) to best mimic the thermalbehavior of the substrate. The reflective surface on the mirror (1770)can be on the top surface or on the bottom surface. In some embodiments,the mirror (1770) is placed on the same substrate (1702) as the reactionvolumes. In some embodiments areas on the substrate can be made to actas mirrors to provide multiple measurement locations on the substrate(1702). An optical assembly, consisting of a source (1771), opticaltrain (1772), and a detector (1774) can be placed outside of thechamber's volume (1709). In some embodiments, measure of the fogging orcondensation of water fine water droplets onto a mirror is used tomeasure the condensation or the evaporation rate. Condensation conditionon the mirror (1770) can be detected by measuring the intensity of theoptical beam (1773) reflected off the surface. The beam (1773) can bemodulated in time and wavelength, via the modulation of the source(1771) to achieve higher signal to noise ratios. In preferredembodiments, the system comprises a control loop. In an exemplaryembodiment, and as depicted in FIG. 25, the control loop consists of adetector (1774) which feeds measured optical intensities to a signalconditioning circuit (1775). The output of the signal conditioningcircuit (1775) is used by the cold mirror logic (1776) to determine thecondensation state on the mirror, and calculate molar ratio of the mixof gases in the volume (1709) using other inputs such as temperature,pressure etc. The system comprises a temperature sensor (1781), pressuresensor (not shown), and/or any other suitable sensors. The humidity andtemperature logic (1777) determines the actuation of humidifier (1778),dehumidifier (1779), and/or temperature controllers (1780) to effectuatethe desirable conditions determined by the cold mirror logic (1776).

In some embodiments, the reaction volumes (1703) contain the necessaryreagents to allow enzyme mediated biochemical reactions to take placebetween the molecular population inside the reaction volume (e.g.droplet) and the molecular population present on the wetted surface incontact with the reaction volume. One would appreciate that the reactionvolume can be used to carry out a variety of reactions including, but nolimited to, amplification, hybridization, extension, ligation,sequencing, in-vitro transcription, in-vitro translation, or any otherreaction of interest. The molecular population may contain nucleicacids, DNA, RNA, oligonucleotides, proteins, dNTPs, salts, buffercomponents, detergents, and/or any other appropriate component. Thereaction volume may comprises an enzyme, such as a polymerase, a ligase,a CEL1-like endonuclease, a nuclease, mixtures of such enzymes, and/orany other appropriate enzymes. In some embodiments, the products of theenzyme mediated biochemical reaction can include contain nucleic acids,DNA, RNA, oligonucleotides, proteins, labeled nucleic acids, amplifiednucleic acid (e.g. clonal amplification of a selected population ofnucleic acid), assembled nucleic acids etc.

In some aspects of the invention, the reagents in the reaction volumespromote oligonucleotide or polynucleotide assembly. In some embodiments,the reaction volumes may contain two or more populations ofsingle-stranded oligonucleotides having predefined sequences insolution. The populations of oligonucleotides can hybridize to asingle-stranded oligonucleotide attached to the wetted surface therebyforming double-stranded hybrids or duplexes attached to the surface. Insome embodiments, the double-stranded hybrids contain breaks and gaps inthe phosphodiester backbone, formed at the junctions of differentoligonucleotide populations. In some embodiments, a polymerase and dNTPsand other necessary components are added to fill the gaps in thebackbone. In other embodiments, a ligase and other necessary componentsare added to mend breaks in the backbone.

In other embodiments, the reaction volumes may contain two or morepopulations of oligonucleotides in solution, each population ofoligonucleotide having predefined sequence. In some embodiments, theeach population of oligonucleotide has a sequence complementary to thean another population of oligonucleotides. In this manner, thepopulations of oligonucleotides can hybridize to form double strandedhybrids or duplexes in solution. The hybrids may contain breaks and gapsin the phosphodiester backbone, formed at the junctions of differentoligonucleotide populations. In some embodiments, a polymerase and dNTPsand other necessary components are added to fill the gaps in thebackbone. In other embodiments, a ligase and other necessary componentsare added to mend breaks in the backbone.

Aspects of the invention provide methods for the amplification of one ormore single-stranded oligonucleotide on the support. Oligonucleotidesmay be amplified before or after being detached from the support and/oreluted in a droplet. Preferably, the oligonucleotides are amplified onthe solid support. One skilled in the art will appreciate thatoligonucleotides that are synthesized on solid support will comprise aphosphorylated 3′ end or an additional 3′-terminal nucleoside (e.g. T).The 3′-phosphorylated oligonucleotides are not suitable forpolynucleotide assembly as the oligonucleotides cannot be extended bypolymerase. In preferred aspects of the invention, the oligonucleotidesare first amplified and the amplified products are assembled into apolynucleotide. Accordingly, aspect of the invention provides methodswherein a set or subset of oligonucleotides, that are attached to at aset of subset of features of the support, are amplified by locallydelivering sub-microvolumes at addressable discrete features. The term“amplification” means that the number of copies of a nucleic acidfragment is increased. As noted above, the oligonucleotides may be firstsynthesized onto discrete features of the surface, may be deposited onthe substrate or may be deposited on the substrate attached tonanoparticles. In a preferred embodiment, the oligonucleotides arecovalently attached to the surface or to nanoparticles deposited on thesurface. In an exemplary embodiment, locations or features comprisingthe oligonucleotides to be amplified are first selected. In a preferredembodiment, the selected features are in close proximity to each otherson the support. Aqueous solution is then deposited on the selectedfeature thereby forming a droplet comprising hydrated oligonucleotides.One would appreciate that each droplet is separated from the other bysurface tension. In some embodiments, the solution can be water, bufferor a solution promoting enzymatic reactions. In an exemplary embodiment,the solution includes, but is not limited to, a solution promotingprimer extension. For example, the solution may be composed ofoligonucleotides primer(s), nucleotides (dNTPs), buffer, polymerase andcofactors. In other embodiments, the solution is an alkaline denaturingsolution. Yet, in other embodiments, the solution may compriseoligonucleotides such as complementary oligonucleotides.

In some embodiments, oligonucleotides or polynucleotides are amplifiedwithin the droplet by solid phase PCR thereby eluting the amplifiedsequences into the droplet. In other embodiments, oligonucleotides orpolynucleotides are first detached form the solid support and thenamplified. For example, covalently-attached oligonucleotides aretranslated into surface supported DNA molecules through a process ofgaseous cleavage using amine gas. Oligonucleotides can be cleaved withammonia, or other amines, in the gas phase whereby the reagent gas comesinto contact with the oligonucleotide while attached to, or in proximityto, the solid support (see Boal et al., Nucl. Acids Res, 1996,24(15):3115-7), U.S. Pat. Nos. 5,514,789; 5,738,829 and 6,664,388). Inthis process, the covalent bond attaching the oligonucleotides to thesolid support is cleaved by exposing the solid support to the amine gasunder elevated pressure and/or temperature. In some embodiments, thisprocess may be used to “thin” the density of oligonucleotides atspecific features.

One skilled in the art will appreciate that releasing oligonucleotidesfrom the solid support can be achieved by a number of differenttechniques which will depend on the technique used to attach orsynthesize the oligonucleotides on the solid support. Preferably, theoligonucleotides are attached or synthesized via a linker molecule andsubsequently detached and released. In some embodiments, a plurality ofoligonucleotides may be attached or synthesized to the support, cleavedat a cleavable linker site and released in solution. For example, U.S.Pat. No. 7,563,600 discloses a cleavable linker having a succinatemoiety bound to a nucleotide moiety such that the cleavage produces a3′-hydroxy-nucleotide. The succinate moiety is bound to solid supportthrough an ester linkage by reacting the succinate moieties with thehydroxyl on the solid support. US Patent application discloses sulfonylcleavable linkers comprising a linker hydroxyl moiety and a base-labilecleaving moiety. A phosphorous-oxygen bond is formed between phosphorousof the sulfonyl amidite moieties and oxygen of the hydroxyl groups atknown location of the support. In some embodiments, the oligonucleotidesare attached or synthesized using a photo-labile linker (see for exampleTosquellas et al., Nucl. Acids Res., 1998, Vol. 26, pp 2069-2074). Insome instances, the photolabile linker can be rendered labile byactivation under an appropriate chemical treatment. For example, U.S.Pat. No. 7,183,406 discloses a safety-catch linker which is stable underthe oligonucleotide synthesis conditions and that is photolabile aftertreatment with trifluoroacetic acid. Oligonucleotides linked with aphoto-labile linker can then be released by photolysis. Usingphotolabile linkers, it is therefore possible to selectively release insolution (e.g. in a droplet) specific oligonucleotides at predeterminedfeatures. The oligonucleotides released in solution may then be broughtinto contact for further processing (hybridization, extension, assembly,etc. . . . ) by merging droplets of moving the oligonucleotides from onefeature to a next feature on a solid support.

One skilled in the art will appreciate that DNA microarrays can havevery high density of oligonucleotides on the surface (approximately 108molecules per feature), which can generate steric hindrance topolymerases needed for PCR. Theoretically, the oligonucleotides aregenerally spaced apart by about 2 nm to about 6 nm. For polymerases, atypical 6-subunit enzyme can have a diameter of about 12 nm. Thereforethe support may need to be custom treated to address the surface densityissue such that the spacing of surface-attached oligonucleotides canaccommodate the physical dimension of the enzyme. For example, a subsetof the oligonucleotides can be chemically or enzymatically cleaved, orphysically removed from the microarray. Other methods can also be usedto modify the oligonucleotides such that when primers are applied andannealed to the oligonucleotides, at least some 3′ hydroxyl groups ofthe primers (start of DNA synthesis) are accessible by polymerase. Thenumber of accessible 3′ hydroxyl groups per spot can be stochastic orfixed. For example, the primers, once annealed, can be treated to removesome active 3′ hydroxyl groups, leaving a stochastic number of 3′hydroxyl groups that can be subject to chain extension reactions. Inanother example, a large linker molecule (e.g., a concatamer) can beused such that one and only one start of synthesis is available perspot, or in a subset of the oligonucleotides per spot.

According to some aspects of the invention, hydrated oligonucleotidescan be amplified within the droplet, the droplet acting as a virtualreaction chamber. In some embodiments, the entire support or arraycontaining the discrete features is subjected to amplification. In otherembodiments, one or more discrete features are subjected toamplification. Amplification of selected independent features (beingseparated from each others) can be performed by locally heating at leastone discrete feature. Discrete features may be locally heated by anymeans known in the art. For example, the discrete features may belocally heated using a laser source of energy that can be controlled ina precise x-y dimension thereby individually modulating the temperatureof a droplet. In another example, the combination of a broader beamlaser with a mask can be used to irradiate specific features. In someembodiments, methods to control temperature on the support so thatenzymatic reactions can take place on a support (PCR, ligation or anyother temperature sensitive reaction) are provided. In some embodiments,a scanning laser is used to control the thermocycling on distinctfeatures on the solid support. The wavelength used can be chosen fromwide spectrum (100 nm to 100,000 nm, i.e. from ultraviolet to infrared).In some embodiments, the feature on which the droplet is spottedcomprises an optical absorber or indicator. In some other embodiments,optical absorbent material can be added on the surface of the droplet.In some embodiments, the solid support is cooled by circulation of airor fluid. The energy to be deposited can be calculated based on theabsorbance behavior. In some embodiments, the temperature of the dropletcan be modeled using thermodynamics. The temperature can be measured byan LCD like material or any other in-situ technology. In someembodiments, the solid support is cooled by circulation of air or fluid.For example, the whole support can be heated and cooled down to allowenzymatic reactions to take place. One method to control the temperatureof the surface droplets is by using a scanning optical energy depositionsetup as shown in FIG. 8. An energy source (406) can be directed by ascanning setup 407 to deposit energy at various locations on the surfaceof the solid support 401 comprising attached or supported molecules(404). Optical absorbent material (402, 405) can be added on the surfaceof the solid support or on the surface of droplet. Optical energysource, such as a high intensity lamp, laser, or other electromagneticenergy source (including microwave) can be used. The temperature of thedifferent reaction sites (420, 421, 422, 423, . . . ) can be controlledindependently by controlling the energy deposited at each of thefeatures.

For example, a Digital Micromirror Device (DMD) can be used fortemperature control. DMD is an optical semiconductor. See, for example,U.S. Pat. No. 7,498,176. In some embodiments, a DMD can be used toprecisely heat selected features or droplets on the solid support. TheDMD can be a chip having on its surface several hundred thousandmicroscopic mirrors arranged in a rectangular array which correspond tothe features or droplets to be heated. The mirrors can be individuallyrotated (e.g., ±10-12°), to an on or off state. In the on state, lightfrom a light source (e.g., a bulb) is reflected onto the solid supportto heat the selected spots or droplets. In the off state, the light isdirected elsewhere (e.g., onto a heatsink). In one example, the DMD canconsist of a 1024×768 array of 16 μm wide micromirrors. These mirrorscan be individually addressable and can be used to create any givenpattern or arrangement in heating different features on the solidsupport. The features can also be heated to different temperatures,e.g., by providing different wavelengths for individual spots, and/orcontrolling time of irradiation.

One would appreciate that amplification occurs only on featurescomprising hydrated template oligonucleotides (i.e. local amplificationat features comprising a droplet volume). Different set of features maybe amplified in a parallel or sequential fashion with parallel orsequential rounds of hydrating (i.e. dispensing a droplet volume on aspecific feature), amplifying oligonucleotides and drying the set offeatures. In some embodiments, the support is dried by evaporatingliquid in a vacuum while heating. Thus, after each round ofamplification, the support will comprise a set of droplets containingoligonucleotides duplexes. The complementary oligonucleotides can bereleased in solution within the droplet and be collected. Alternatively,complementary oligonucleotides may be dried onto the discrete featuresfor storage or further processing. Yet, complementary oligonucleotidescan be subjected to further reactions such as error filtration and/orassembly. In some embodiments, a different set or subset of features canthen be hydrated and a different set or subset of templateoligonucleotides can be amplified as described herein. For example, adroplet 810, as illustrated in FIG. 9, can be dispensed (e.g.,inkjetted) on a support 800. The droplet 810 can contain variousreagents such as enzymes, buffers, dNTPs, primers, etc. The droplet 810covers a discrete feature 820 (a feature corresponding to a predefinedsequence) on the support 800. For purpose of illustration only, fouroligonucleotides, 801, 802, 803, 804 are shown, while many moreoligonucleotides having the same sequence are also present on feature820 but not shown. PCR can be carried out to synthesize oligonucleotides801′, 802′, 803′, 804′ complementary to template oligonucleotides 801,802, 803, 804 that are attached to feature 820. In the case of theenzymatic amplification, the solution includes but is not limited toprimers, nucleotides, buffers, cofactors, and enzyme. For example, anamplification reaction includes DNA polymerase, nucleotides (e.g. dATP,dCTP, dTTP, dGTP), primers and buffer.

In some embodiments, a selected set of features may be protected fromhydration by using an immiscible fluid system. An immiscible fluidsystem, such as oil and aqueous reagents, can be used to achievepassivation of sites on which reactions take place. As shown in FIG. 22,a droplet of oil (or a short chain hydrocarbon) can first be depositedon a site (403) where reaction is undesirable. After the oil deposition,subsequent fluid processing steps will affect only the unprotected sitesor features (404), but not the protected sites or features (403) sincethe fluid (402), cannot reach the surface of the protected site (403).This concept can be further extended to allow controlled exposure orprotection at the oil covered spots (403). By using electrowettingconcepts, the shape of an oil droplet can be modulated by theappropriate application of electric field. The surface droplet shape canbe modulated from its normal state (409) to its actuated state (410) byelectrowetting or optoelectrowetting. The effect of such control allowsthe exposure of a portion or the totality of the feature (109) dependingon the applied field.

In some embodiments, the oligonucleotides may comprise universal (commonto all oligonucleotides), semi-universal (common to at least of portionof the oligonucleotides) or individual or unique primer (specific toeach oligonucleotide) binding sites on either the 5′ end or the 3′ endor both. As used herein, the term “universal” primer or primer bindingsite means that a sequence used to amplify the oligonucleotide is commonto all oligonucleotides such that all such oligonucleotides can beamplified using a single set of universal primers. In othercircumstances, an oligonucleotide contains a unique primer binding site.As used herein, the term “unique primer binding site” refers to a set ofprimer recognition sequences that selectively amplifies a subset ofoligonucleotides. In yet other circumstances, an oligonucleotidecontains both universal and unique amplification sequences, which canoptionally be used sequentially.

In some embodiments, primers/primer binding site may be designed to betemporary. For example, temporary primers may be removed by chemical,light based or enzymatic cleavage. For example, primers/primer bindingsites may be designed to include a restriction endonuclease cleavagesite. In an exemplary embodiment, a primer/primer binding site containsa binding and/or cleavage site for a type IIs restriction endonuclease.In such case, amplification sequences may be designed so that once adesired set of oligonucleotides is amplified to a sufficient amount, itcan then be cleaved by the use of an appropriate type IIs restrictionenzyme that recognizes an internal type IIs restriction enzyme sequenceof the oligonucleotide. In some embodiments, after amplification, thepool of nucleic acids may be contacted with one or more endonucleases toproduce double-stranded breaks thereby removing the primers/primerbinding sites. In certain embodiments, the forward and reverse primersmay be removed by the same or different restriction endonucleases. Anytype of restriction endonuclease may be used to remove theprimers/primer binding sites from nucleic acid sequences. A wide varietyof restriction endonucleases having specific binding and/or cleavagesites are commercially available, for example, from New England Biolabs(Beverly, Mass.). In various embodiments, restriction endonucleases thatproduce 3′ overhangs, 5′ overhangs or blunt ends may be used. When usinga restriction endonuclease that produces an overhang, an exonuclease(e.g., RecJf, Exonuclease 1, Exonuclease T, S1 nuclease, P1 nuclease,mung bean nuclease, T4 DNA polymerase, CEL I nuclease, etc.) may be usedto produce blunt ends. Alternatively, the sticky ends formed by thespecific restriction endonuclease may be used to facilitate assembly ofsubassemblies in a desired arrangement. In an exemplary embodiment, aprimer/primer binding site that contains a binding and/or cleavage sitefor a type IIs restriction endonuclease may be used to remove thetemporary primer. The term “type-Hs restriction endonuclease” refers toa restriction endonuclease having a non-palindromic recognition sequenceand a cleavage site that occurs outside of the recognition site (e.g.,from 0 to about 20 nucleotides distal to the recognition site). Type Hsrestriction endonucleases may create a nick in a double-stranded nucleicacid molecule or may create a double-stranded break that produces eitherblunt or sticky ends (e.g., either 5′ or 3′ overhangs). Examples of TypeHs endonucleases include, for example, enzymes that produce a 3′overhang, such as, for example, Bsr I, Bsm I, BstF5 I, BsrD I, Bts I,Mnl I, BciV I, Hph I, Mbo II, Eci I, Acu I, Bpm I, Mme I, BsaX I, Bcg I,Bae I, Bfi I, TspDT I, TspGW I, Taq II, Eco57 I, Eco57M I, Gsu I, Ppi I,and Psr I; enzymes that produce a 5′ overhang such as, for example, BsmAI, Ple I, Fau I, Sap I, BspM I, SfaN I, Hga I, Bvb I, Fok I, BceA I,BsmF I, Ksp632 I, Eco31 I, Esp3 I, Aar I; and enzymes that produce ablunt end, such as, for example, Mly I and Btr I. Type-Hs endonucleasesare commercially available and are well known in the art (New EnglandBiolabs, Beverly, Mass.).

After amplification, the polymerase may be deactivated to preventinterference with the subsequent steps. A heating step (e.g. hightemperature) can denature and deactivate most enzymes which are notthermally stable. Enzymes may be deactivated in presence (e.g. withinthe droplet) or in the absence of liquid (e.g. dry array). Heatdeactivation on a dry support has the advantage to deactivate theenzymes without any detrimental effect on the oligonucleotides. In someembodiments, a non-thermal stable version of the thermally stable PCRDNA Polymerase may be used, although the enzyme is less optimized forerror rate and speed. Alternatively, Epoxy dATP can be use to inactivatethe enzyme.

In some embodiments, discrete features may contain oligonucleotides thatare substantially complementary (e.g. 50%, 60%, 70%, 80%, 90%, 95%, 98%,99% or 100%). With reference to FIG. 9, template oligonucleotides 801,802, 803, 804 can have inherent errors as they are generally chemicallysynthesized (e.g., deletions at a rate of 1 in 100 bases and mismatchesand insertions at about 1 in 400 bases). Assuming an average error rateof 1 in 300 bases and an average template oligonucleotide size of 70bases, every 1 in 4 template oligonucleotides will contain an errorcompared to a reference sequence (e.g., the wide-type sequence of a geneof interest). For example, referring to FIG. 9, template oligonucleotide803 contains an error 805 which can be a mismatch, deletion, orinsertion. During PCR synthesis, the error 805 is retained in thesynthesized oligonucleotide 803′ as error 805′. Additional errors (notshown) can be introduced during PCR. Methods for error correction areneeded for high-fidelity gene synthesis/assembly.

In one embodiment, error-containing oligonucleotides are removed by amethod illustrated in FIG. 9. PCR products comprising oligonucleotidesduplexes (e.g. duplexes 801-801′, 802-802′, 803-803′, 804-804′) aredenatured by melting the duplexes (e.g., at elevated temperature, usinga helicase, etc.), forming free oligonucleotides 801′, 802′, 803′, 804′.In some embodiments, using a helicase to melt duplexes can provide forisothermal denaturing without elevating the temperature. The term“duplex” refers to a nucleic acid molecule that is at least partiallydouble-stranded. A “stable duplex” refers to a duplex that is relativelymore likely to remain hybridized to a complementary sequence under agiven set of hybridization conditions. In an exemplary embodiment, astable duplex refers to a duplex that does not contain a basepairmismatch, insertion, or deletion. An “unstable duplex” refers to aduplex that is relatively less likely to remain hybridized to acomplementary sequence under a given set of hybridization conditionssuch as stringent melt. In an exemplary embodiment, an unstable duplexrefers to a duplex that contains at least one base-pair mismatch,insertion, or deletion.

Next, under annealing conditions (e.g., lower temperature),oligonucleotides 801′, 802′, 803′, 804′ will randomly anneal to templateoligonucleotides 801, 802, 803, 804. By way of example, new duplexes801-803′, 802-801′, 803-804′ and 804-802′ can be formed. 802-801′ and804-802′ are error-free duplexes, or stable duplexes whereas 801-803′and 803-804′ each contain a mismatch between the two complementarystrands (unstable duplexes). All duplexes within a feature are thensubject to a stringent melting step to denature 801-803′ and 803-804′,leaving 802-801′ and 804-802′ intact. Oligonucleotides 803′ (containingerror 805′) and 804′ can then be removed or washed away. Error-freeoligonucleotides 801′ and 802′ can be melted and recovered in a dropletfor subsequent amplification, ligation, and/or chain extension. Thesesteps can be repeated multiple times to enrich for error-freeoligonucleotides, as support 800 can be washed and reused at leastseveral times. Repeating the steps of denaturing and annealing allowsthe error-containing oligonucleotides to partner with differentcomplementary oligonucleotides within the droplet, producing differentmismatch duplexes. These can also be detected and removed as above,allowing for further enrichment for the error-free duplexes. Multiplecycles of this process can in principle reduce errors to undetectablelevels. Local removal of error-containing oligonucleotides can berepeated at different features in a sequential fashion by drying thesupport between each different stringent melt conditions.

The conditions for stringent melt (e.g., a precise melting temperature)can be determined by observing a real-time melt curve. In an exemplarymelt curve analysis, PCR products are slowly heated in the presence ofdouble-stranded DNA (dsDNA) specific fluorescent dyes (e.g., SYBR Green,LCGreen, SYTO9 or EvaGreen). With increasing temperature the dsDNAdenatures (melts), releasing the fluorescent dye with a resultantdecrease in the fluorescent signal. The temperature at which dsDNA meltsis determined by factors such as nucleotide sequence, DNA length andGC/AT ratio. Typically, G-C base pairs in a duplex are estimated tocontribute about 3° C. to the Tm, while A-T base pairs are estimated tocontribute about 2° C., up to a theoretical maximum of about 80-100° C.However, more sophisticated models of Tm are available and may be inwhich G-C stacking interactions, solvent effects, the desired assaytemperature and the like are taken into account. Melt curve analysis candetect a single base difference. Various methods for accuratetemperature control at individual features can be used as disclosedabove.

In some embodiments, the entire support or array containing the discretefeatures is heated to a denaturing temperature. Preferably, denaturationof double stranded nucleic acid is performed in solution (e.g. withinthe droplet). During the heat denaturation step, the temperature of thesupport is raised to a stringent melt temperature or to a denaturingtemperature (95° C. to 100° C.). Elevating the temperature of thesupport to a denaturing or stringent melt temperature allows thehomoduplexes to dissociate into single strands before completeevaporation of the droplet volume. Heating the substrate results in thedenaturation and evaporation of the solution, resulting in driedsingle-stranded oligonucleotides onto the discrete features. At thispoint, the entire support may be cooled down to a predefinedhybridization or annealing temperature. A set of selected features orthe totality of the features may be re-hydrated by addition of theappropriate annealing buffer (at the appropriate annealing temperature)at the selected features or on the entire support. Single strandedoligonucleotides may then be resuspended and allowed to diffuse and tohybridize or anneal to form the double-stranded oligonucleotides(homoduplexes or heteroduplexes).

Accordingly, some aspects of the invention relate to the recognition andlocal removal of double-stranded oligonucleotides containing sequencemismatch errors at specific features. In one preferred embodiment of theinvention, mismatch recognition can be used to control the errorsgenerated during oligonucleotide synthesis, gene assembly, and theconstruction of longer polynucleotides. After amplification, thetotality of the features or a set of the features comprisingoligonucleotide duplexes are first subjected to round(s) of melting andannealing as described above (FIG. 10). Subsequently, a first set ofdiscrete features comprising oligonucleotides having same theoretical Tmare hydrated and oligonucleotides are allowed to anneal under annealingconditions. Hydrated features are then subjected to a first stringentmelt condition (condition 1). It would be appreciate that for sequentiallocal error removal, it is preferable to first start with the stringentmelt conditions corresponding to the lowest Tm (Tm(1)) and conclude withstringent melt conditions corresponding to the higher Tm (Tm(n)). Inother embodiments, the totality of the features of the support may behydrated and subjected to the lowest Tm temperature. Under the firstspecific stringent melt conditions Tm(1), only the oligonucleotides thatare hybridized in an unstable duplex will de-hybridize (see FIG. 10A).De-hybridized oligonucleotides may be removed for example, using avacuum or may be washed away. In a subsequent step, the support may bedried out and a second discrete features comprising oligonucleotideshaving a Tm higher than Tm(1) (for example (Tm(2)) are selectivelyrehydrated and allowed to anneal under annealing conditions. In otherembodiments, the totality of the features of the support may bere-hydrated and subjected to the second Tm temperature Tm(2) whereinTm(2) is higher than Tm(1) (FIG. 10B). These steps of selectivehydration, annealing, stringent melt and removal of error-containingoligonucleotides can be repeated multiple times until all discretefeatures have been subjected to the appropriate stringent melt condition(theoretically 80-100° C.). Alternatively, a mismatch detectingendonuclease may be added to the droplet solution. In an exemplaryembodiment, a Surveyor™ Nuclease (Transgenomic Inc.) may be added to thehydrated feature containing the oligonucleotide duplexes. Surveyor™Nuclease is a mismatch specific endonuclease that cleaves all types ofmismatches such as single nucleotide polymorphisms, small insertions ordeletions. Addition of the endonuclease results in the cleavage of thedouble-stranded oligonucleotides at the site of the mismatch. Theremaining portion of the oligonucleotide duplexes can then be melted ata lower and less stringent temperature (e.g. stringent melt) needed todistinguish a single base mismatch. One would appreciate that the errorremoval steps as well as the amplification steps may be repeated in asequential fashion or in a highly parallel fashion by controlling thetemperature of the entire support or of the independent features asdescribed above.

In a preferred aspect of the invention, oligonucleotides havingpredefined sequences are assembled after being amplified anderror-filtered. In some embodiments, two adjacent droplets containingtwo multiple copies of different oligonucleotides or polynucleotides insolution are combined by merging the appropriate droplets on the solidsupport as illustrated in FIG. 11 and FIG. 12. In FIG. 12, the solidsupport comprises different and unique molecules (201, 202, 203, 204)supported or attached to the surface of 102, a unique molecule (250)supported or attached to the surface of 102 at multiple positions andother unique molecules (299) supported or attached to the surface of102. On the solid support surface (102) an existing pattern of moleculescan be found. For example, as illustrated in FIG. 12, differentmolecules or oligonucleotides can exist at different positions, as shownby the placement of 201, 202, 203, 204, 250, and 299. One shouldappreciate that the arrangement of these unique molecules (201, 202,203, 204) can be designed to strategically allow the subsequentcombining of the contents of these sites. For example, these uniquemolecules can be arranged in a checker board pattern. The first checkerboard pattern contains 201 and 202. After individual reactions withinthe microvolume of 201 and 202 are complete, the user can choose tocombine the content of 201 and 202 by forming a droplet that encompassesboth 201 and 202 sites. In another subsequent step, the content of201+202 can be combined with the content of 203+204, to form a reactionthat contains all four reaction products of the unique molecules (201,202, 203, 204). FIG. 11A illustrates the same general concept with A, B,C, D. In step 0, all the unique molecules are reacted in separatevolumes. In Step 1, the adjacent sites are combined, to give A+B, andC+D, etc. In Step 2, A+B can be combined with C+D, and etc. In Step 3,another level of aggregation is added. One should appreciate that thereis no limit to the number of steps that can be implemented. In FIG. 11B,two possible arrangement strategies are outlined. In the first strategy,some adjacent sites comprise the same molecules or oligonucleotides(e.g. A and B) and the four sites may be combined to generate a circulardroplet. In the second strategy, each site comprises a unique anddifferent molecule or oligonucleotide and each site can be combined toan adjacent site.

For example, with reference to FIG. 12, solid supported oligonucleotide201 and oligonucleotides 202 may be amplified in first stage droplet 1and first stage droplet 2. After amplification, each first stage dropletcontains one amplified double-stranded oligonucleotide sequence. Inembodiments, multiple copies of oligonucleotides 201 and multiple copiesof oligonucleotide 202 are eluted within the first stage droplet 1 andthe first stage droplet 2, respectively. The two first stage dropletsbeing in close proximity to each other are combined to form a secondstage droplet. In some embodiments, two different or moreoligonucleotides or polynucleotides may be immobilized or synthesized atthe same location (or feature) on the solid support thereby facilitatingtheir interaction after amplification within the same droplet. See e.g.US 2004/0101894. In some embodiments, droplets are merged to form biggerdroplets by adding, or spotting additional “merger” droplets or volumesin between or around the appropriate original droplets. Two droplets, orisolated volumes can therefore merge if a “merger” droplet or volume iscreated and expanded until the merge takes place. The resultant mergedvolume will encompass the first stage droplets or first isolatedvolumes. The volume and location of the resulting merged volume canvary. The merged volumes (e.g. second stage droplet) can occupy afootprint that is the combination of all volumes (e.g. first stagedroplets and merger droplet). Alternatively, the merged volumes canoccupy at least part of the footprint of one of the isolated volume(e.g. first or second isolated volume.

Some aspects of the invention, relate to the destination selection androuting of the isolated volumes and therefore to the control of thelocation or footprint of merged volumes. One would appreciate that asindividual regions of the support are addressable, individual isolatedvolumes such as droplets may be controlled individually. In someembodiments, it is preferable to place isolated volumes onto adjacentregions or features to allow merging of the volumes. Yet, in otherembodiments, isolated volumes are directed or routed to a pre-selecteddestination.). In some case, the merged volumes occupy the footprint ofone of the isolated volume and extend to one or more smaller contactangle regions (SCA). In some embodiments, the substrate of the supportis substantially planar and droplets are routed using a two-dimensionalpath (e.g. x,y axis). Droplets may be moved to bring them to selectedlocations for further processing, to be merged with a second isolatedvolume into a second stage droplet at preselected locations and/orduring the transport, to remove some reactants from the droplet(referred herein “wash-in-transport’ process).

In some embodiments, step-wise hierarchical and/or sequential assemblycan be used to assemble oligonucleotides and longer polynucleotides. Ina preferred embodiment, the methods use hierarchical assembly of two ormore oligonucleotides or two or more nucleic acids subassemblies at atime. Neighboring droplets can be manipulated (move and/or merged, asdescribed above) to merge following a hierarchical strategy therebyimproving assembly efficiency. In some embodiments, each dropletcontains oligonucleotides with predefined and different nucleic acidsequences. In some embodiments, two droplets are moved following apredefined path to an oligonucleotide-free position. In a preferredembodiment, the assembly molecules (e.g. oligonucleotides) arepre-arranged on the support surface at pre-determined discrete features.For example, with reference to FIG. 13A, the support comprises differentunique molecules (e.g. oligonucleotides S1 to Sn). The arrangement ofthe molecules is designed to strategically allow the hierarchicalassembly of S1 with S2, S2 with S4 etc. . . . by manipulatingneighboring droplets S1 and S2, S3 and S4, and merge the droplets toform the S1-2, S3-4, S(n−1)-(n) first stage droplets. In someembodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more droplets may be combinedat each step. In a preferred embodiment, droplets (S1, S2, . . . ) aremoved and combined in an oligonucleotide-free position or feature toform first stage droplets (S1,−2, S3,−4, S(n−1),). In a second step,another level of aggregation is added. For example, referring to FIG.13A, first stage droplet S1,2 can be combined to first stage dropletS3,4 to form a merged second stage droplet S1,2,3,4. The hierarchicalassembly terminal merging step results in droplet S1,2,3, . . . ,ncontaining the content of droplet S1+S2+ . . . Sn. Furthermore,different terminal merging location (two or more assembly groups) can bestrategically positioned next to each other to allow for the combinationof different assembly droplets and thereby for the assembly of apredetermined sequence (FIG. 13B). In some embodiments, 2, 3, 4, 5, 6,7, 8, 9, 10 or more assembly groups can be arranged to minimize droplettravel distance to form the predetermined sequence. In some embodiments,the assembly members within each group are arranged on the supportsurface in a pattern minimizing the droplet travel distance required forthe formation of the M1,2,3 . . . n, Q1,2,3 . . . n, R1,2,3 . . . n,S1,2,3 . . . n products. In a further embodiments, the position of theM1,2,3 . . . n, Q1,2,3 . . . n, R1,2,3 . . . n, S1,2,3 . . . n arearranged on the support surface in a pattern minimizing the droplettravel distance required for the formation MQRS(1,2,3 . . . n).

One should appreciate that isolated volumes may be routed independentlyin a sequential or highly parallel fashion. Droplets may be routed usingelectrowetting-based techniques (see for example, U.S. Pat. No.6,911,132 and U.S. Patent Application 2006/0054503). Electrowettingprinciple is based on manipulating droplets on a surface comprising anarray of electrodes and using voltage to change the interfacial tension.By applying an electric field (e.g. alternating or direct), the contactangle between the fluid and surfaces can be modified. For example, byapplying a voltage, the wetting properties of a hydrophobic surface canbecome increasingly hydrophilic and therefore wettable. In someembodiments, the array of electrode is not in direct contact with thefluid. In some embodiments, droplets are moved using a wettabilitygradient. It has been shown that droplets placed on wettability gradientsurfaces typically move in the direction of increasing wettability (seeZielke and Szymczyk, Eur. Phys. J. Special Topics, 166, 155-158 (2009)).In other embodiments, droplets may be moved using a thermal gradient.When placed on a thermal gradient, droplets move from higher temperaturelocations towards lower temperature locations. Moving droplets usingelectrowetting, temperature gradients and wettability gradients dependon the liquid (e.g. aqueous, non-aqueous, solute concentration), thesize of the droplets and/or the steepness of the gradient.

One skilled in the art will appreciate that most of the electrowettingmerging and mixing strategies rely on the fact that droplets haveidentical volumes before merging. In some aspects of the invention,routing of the droplet and merging is controlled by the using differentsize droplets. In a preferred embodiment, the footprint of the mergedvolume is controlled by the size of the droplets before merging. In someembodiments, the method comprises moving the content of smaller volumedroplets to the position of arger volume droplets. Referring to FIG. 3,two isolated volumes or droplets (31, 32) are created, the volume ofdroplet 31 being smaller than the volume of droplet 32. The droplets areseparated from each other by a region 39. Region 39 may comprise atleast one SCA, at least one HCA, or any combination of SCA and HCA.After a merger droplet 33 is created, it may contact droplet 31 ordroplet 32 or both droplet 31 and droplet 32. The resultant volumeoccupies the footprint or a substantial part of the footprint of thelarger of the two original isolated liquid volumes. For example, asillustrated in FIG. 3, the merged volume 34A can occupy the footprint ofthe larger droplet 32 or the merged volume 34B can occupy a substantialpart of the footprint of the larger droplet.

One skilled in the art will appreciate that the principle describedherein can be applied to move liquid volumes such as droplets on thesupport along a predetermined path and to determine the exact locationof the merged volume. In some embodiments, the content of the smallervolumes may repeatedly be moved to the position of larger volumes inorder to move liquid volumes over a distance that is larger than amerger region. The principle is illustrated in FIG. 4 and FIG. 5. In anexemplary embodiment, a first isolated volume referred as “solutiondroplet” is created (e.g solution droplet 41, FIG. 4A; solution droplet410, FIG. 4B, solution droplet 51, FIG. 5). The solution typicallycontains molecules of interest (e.g. oligonucleotides) or solute ofinterest. In some embodiments, a group of droplets (411, FIG. 4B) isadded to a position close to the leading edge of the solution droplet inthe direction of the intended droplet displacement or path. The wettingof the leading edge of the solution droplet 410, together with thedynamic forces caused by the deposition of the group of droplets 411causes the solution droplet to move in the direction of the leadingedge, thereby resulting in the displacement of the solution droplet 410to form droplet 412 at a new position on the support. This process canbe repeated to allow the displacement of the solution droplet to thedesired location on the support. In some embodiments, the volume of thedroplet 412 may be adjusted by evaporation, thereby allowing the dropletto return to its original volume (410), before repeating the procedure.In other embodiments, a solution droplet is created (41, FIG. 4A, 51,FIG. 5). Simultaneously or subsequently, a second isolated volumereferred as an “anchor droplet” is created in the direction of theintended “solution droplet” path or route. A third volume referred as“merger droplet” (43, FIG. 4) is created between the first and secondisolated volumes. Once merged, the resultant volume (44, FIG. 4)occupies the location of the anchor droplet (42, FIG. 4). In someembodiments, the solution droplet and the anchor droplets contain thesolutes and/or molecules of interest. Yet in other embodiment, thesolution droplet contains the solute and/or the molecules of interest.In some embodiments, the merger droplet contains water. Yet in otherembodiments, the merger droplet contains solute and/or molecules ofinterest. Referring to FIG. 6, a solution droplet 61 containing thesolute of interest is inkjetted on to the substrate at position orfeature 600 (FIG. 6, step 1). Subsequently, an anchor droplet, forexample water or buffer, is inkjetted onto the substrate at position orfeature 601, wherein feature 601 is in the direction of the droplet path(FIG. 6, step 2). A merger droplet 63 is then injected between thesolution and the anchor droplet (FIG. 6, step 3). The solution droplet,merger droplet and anchor droplet are merged and solutes are mixed byeither passive diffusion or active mixing. The resultant volume 64,referred as the “merged droplet”, occupies the footprint of the largeranchor droplet (FIG. 6, step 5). After evaporation, the merged dropletreturns to the original size of the original solution droplet (FIG. 6,step 6). In some embodiments, the resultant volumes (merged droplets)are adjusted by allowing evaporation (to reduce its size) or by addingfluid (to increase its size) and can be moved by repeating steps 1through 6. As illustrated in FIG. 4, after adjustment of the size of themerged droplet (44 to 44A), another isolated volume (45) is placedadjacent to (44) in the direction of the intended droplet move. Usingthe same merging process described above, a merger droplet 46 is addedresulting a merged volume 47. These steps can be repeated until thepayload solute originally in droplet 41 is moved to a desirable orpre-selected location. One or more of theses newly merged droplets maybe manipulated according the same protocol which includes deposition ofan anchor droplet, deposition of a merger droplet, merging and mixing,and evaporation. In some embodiments, as illustrated in FIG. 6, the“solution” droplet 61 is diluted by the “merge” droplet 63 and the“anchor” droplet 62 and is then re-concentrated by evaporation of thedroplet 65. Merged droplets and reaction features act as virtual wellsor chambers in which the reactions take place. Reactions, include, butare not limited to incubation, enzymatic reactions, dilution, mixing,error reduction and/or assembly. Although the figures show a linear, onedimensional, path, it should be appreciated that the droplet can bemoved anywhere on the support surface. In some embodiments, the dropletsare moved in a two dimensional direction. Any other operations derivedform this protocol can be envisioned. For example, droplets can bedeposited sequentially, simultaneously, or in a parallel fashion.Droplets may contain only water and may be used as dilution droplets.Other droplets may contain a solute. Droplet content may be mixed bypassive diffusion or active mixing. In some embodiments, at least twodroplets are moved independently following a similar path and are thenmoved towards a feature that is referred as a reaction feature whereinthe droplets are merged. The first and second droplet paths across thesubstrate may follow the same direction or may follow oppositedirections. For example, a first droplet may be moved toward astationary second droplet or the first and the second droplet may bemoved toward each others. Moreover, if two droplets have the same size,reduction of the size of one droplet will enable it to move in thedirection and to the location of the larger volume. Reduction of thesize of the droplet can be achieved by evaporation. Evaporation ofliquid may be achieved using any technique known in the art. Forexample, the isolated liquid volume to be decreased may be heated toinduce or accelerate evaporation. Alternatively, to merge a firstdroplet at the location of a second droplet, liquid may be added to thesecond droplet to increase its size comparatively to the first droplet.

Another benefit of the droplet movement process described herein is theimplementation of a “wash” operation (referred herein aswash-in-transportation). The movement of the liquid away from a surfacefeature allows the separation of the surface-bound molecules (e.g.oligonucleotides) from the molecules in solution. Hence, a washoperation is therefore implemented. For example, wash-in-transportationcan be used to remove the template oligonucleotides form thecomplementary oligonucleotides after amplification. In some embodiments,“wash-in transportation” features or wash spots may be placed adjacentto features where oligonucleotide processing takes place. Referring toFIG. 14, S1, S2, S3, and S4 (602) represent features carryingoligonucleotides or nucleic acid assembly components. Wash spots W1, W2,W3, and W4 (601) are placed adjacent to the S1, S2, S3, and S4 features.Undesirable products released in the droplet solution on features S1,S2, S3, and/or S4 can be moved to the wash spots features W1, W2, W3,and W4, respectively. In some embodiments, the support provides one washspot W for each assembly feature S or a common wash spot for two or moreassembly features. Wash-in-transportation process can also be used toremove unwanted error-containing oligonucleotides from stable duplexesafter annealing and stringent melt. For example, referring to FIG. 14,“stringent melt” features SM (603) can be placed along the path ofnucleic acid assembly progression, allowing for stringent melt errorcorrection as described above. Similarly, the support may comprise oneSM spot for each assembly step or a common SM spot for two or moreassembly features.

In some embodiments, the “merger” droplets or the “anchor” droplet maycontain or not contain enzyme (e.g. polymerase, ligase, etc.),additional oligonucleotides and all reagents to allow assembly by PCR orby ligation (enzymatic or chemical) or by any combination of enzymaticreaction. For example, oligonucleotides in a given droplet may hybridizeto each other and may assemble by PCR or ligation. The bigger dropletsor second stage droplets contain polynucleotides subassemblies and canbe subsequently merged to form larger droplets or third stage dropletcontaining larger fragments. As used herein the term subassembly refersto a nucleic acid molecule that has been assembled from a set ofoligonucleotides. Preferably, a subassembly is at least 2-fold or morelong than the oligonucleotides. For example, a subassembly may be about100, 200, 300, 400, 500, 600, or ore bases long. One should appreciatethat the use of droplets as isolated reaction volumes enables a highlyparallel system. In some embodiments, at least 100, at least 1,000reactions can take place in parallel. In some embodiments, the primersare immobilized on the support in close proximity to the spotscontaining the oligonucleotides to be assembled. In some embodiments,the primers are cleaved in situ. In some embodiments, the primers aresupported on the solid support. The primers may then be cleaved in situand eluted within a droplet that will subsequently merged with a dropletcontaining solid supported or eluted oligonucleotides.

Some aspects of the invention relate to the droplet-based liquidhandling and manipulation methods by implementing electrowetting-basedtechniques. In some embodiments, the microfluidics device comprises twoprimary supports or substrates (FIG. 15, elements (101) and (102)). Eachsubstrate has features on one or both sides to implement the necessaryelectrodes (FIG. 15: 103: control electrodes, 105: bottom sideelectrode, 106: top side electrode) as well as insulation and surfacemodifications (FIG. 15: 104, 107). In an exemplary embodiment, theinsulation and/or surface modification layer is a dielectric and hascontrolled surface quality such that it is hydrophobic or hydrophilic.In some embodiments, the substrate (102) has the top side electrode(106) and/or the bottom side electrode (105). In the case of a substratehaving only the top side electrode, the substrate (102) is configured asshown in FIG. 15 B. In the case of a substrate having only a bottomelectrode, the substrate (102) is configured as shown in FIG. 15C.

In a preferred embodiment, the substrate (102) is a solid support orsurface (108). One should appreciate that a variety of molecules such asoligonucleotides, nucleic acids, peptides, proteins (e.g. antibodies),polysaccharides, etc. . . . may be attached to the support as disclosedherein (see FIG. 16A) Preferably, a library of molecules are attached adifferent location of the support. For example, members of this library(109) may be attached to the substrate via covalent bonding, Van derWaals forces, or any other attachment mechanisms. One should appreciatethat it is possible to manipulate a droplet (110) from one position toanother position (111) to another position (112) without limitation indirection (in both x and y direction, defining a plane) as shown in FIG.16B. While FIG. 16 shows a substrate (102) with top side electrode, FIG.17 shows a substrate (102) with bottom electrodes. In preferredembodiment, methods are provided for the assembly of a polymer such aspolynucleotides from shorter oligonucleotides molecules (see FIG. 18).In some embodiment, the nucleic acid molecule may be assembled and theassembly process may include one or more step describe herein. In anexemplary embodiment, the assembly steps include an extension step (FIG.18 A-C), a shuffling (FIG. 18 D,E), a washing step (FIG. 19 A,B), anerror Correction (FIG. 19 C-E), a length-dependent melt and wash step(FIG. 20), and a merging and assembly step (FIG. 21) or any combinationthereof.

As shown in FIG. 18, individual spots (201, 202) comprise members of ahigh density high diversity library. Individual droplets (203, 204) canbe formed and placed at the locations corresponding to the individualspots (201, 202) via standard electrowetting techniques. As describedabove, the droplet solution may comprise specific enzymes catalyzingbiochemical reactions such as nucleic acid (or polymer) extensionresulting in the extended nucleic acid molecule or polymer (205, 206).In an exemplary embodiment, the members of the library are templateoligonucleotides and the extended products (207, 208) are copies of theoligonucleotides, thereby forming double stranded oligonucleotidesstrands or duplexes. Oligonucleotide strands can dissociate from theirtemplate via thermal or chemical treatments. Oligonucleotides can alsobe detached from the substrate via thermal or chemical treatments. Forexample, nucleic acid copies can be melted off the template byincreasing the temperature of the droplets to above the meltingtemperature of the duplex, as shown in FIG. 18D. Once the droplets areallowed to cool, the copies will re-anneal back to the surface-attachedtemplates. However, since the re-annealing process is random, a naturalshuffling takes place. As shown in FIG. 18C, the duplexes (205) has onetemplate containing an error. The copy from the error containingtemplate, after re-annealing, has a very high probability to hybridizewith a template strand that is not the exact strand where the copy ismade. This shuffling process creates hetero-duplexes (209, 210) that arenot perfectly matching each other, allowing for subsequent recognitionof mismatches in these hetero-duplexes (see copending U.S. Provisionalapplication 61/264,643, filed on Nov. 25, 2009, which is incorporated byreference herein in its entirety). Prior to mismatch recognition, thehetero-duplexes can be washed to remove the previously active extensionenzymes, or to simple change the buffer condition. In this operation,the droplets are forced out via a flow of fluid, either liquid or gas,to purge the contents between substrate (101) and (102), as shown inFIG. 19B. Subsequent to the wash step, a second buffer and enzyme systemor enzymes mixture can be introduced in the same way as before (203,204), for example via standard electrowetting techniques, resulting indroplets (211, 212). In an exemplary embodiment, the second buffer maycomprise an endonuclease that recognizes mismatch or an endonuclease anda DNA ligase. For example, the endonuclease is the CEL1 enzyme whichcleaves heteroduplex DNA at single base-pair mismatch (Surveyor™Nuclease, Transgenomic Inc.). In some embodiments, DNA ligase can beadded to heal non-specific cleavage by the endonucleases (e.g. CEL1).The enzyme (or enzyme mixture), cleaves at the mismatch sites created bythe shuffling step described above, resulting in truncated template-copystubs (214, 216) and cleaved products (213, 215). The cleaved productscan be removed with a second wash step, as shown in FIG. 19E. Anadditional truncation removal step can be performed after the mismatchcleave. A third buffer is introduced and forms droplets (219, 220).Temperature of the droplets is increased to cause the shorter truncatedproducts (221, 222) to melt, while keeping the long full-length duplexesattached to the surface. The temperature can be carefully selected togive very precise discrimination for length and even sequences. A washstep can be carried out to remove the truncated products (221, 222).After wash, the population of polymer still attached to the surface hasenhanced purity and reduced error density (FIG. 20C). In a preferredembodiment, the purified and error-corrected oligonucleotides (or shortpolymers) are utilized in an assembly reaction to create longerpolynucleotides (or longer polymers). For example, as shown in FIG. 20,droplets containing enzymes and suitable buffer can be formed asdescribed before. The droplets (225, 226) each covers a spot thatcontain one or more members of the high density high diversity library.By raising the temperature of the droplets, copies can be release intothe liquid phase or droplet (227, 228). Individual members of thelibrary (or groups of the library) can be combined in a precise andcontrolled manner to create a reaction volume where desirablepopulations (230) of assembly polymers is gathered (229). In a preferredembodiment, the droplet comprises enzymes, such as ligase or polymerase,and the longer polynucleotide may be assembled by polymerase or ligasemediated assembly reaction. The enzyme(s) in the fluid can assemble thesaid population into a desirable longer chain polymer. The assemblysteps described above can be repeated to create increasingly longerpolymer chain or polynucleotide until the desirable product is reached.In some embodiments, droplets are covered with an immiscible solution(e.g. oil) forming a layer over the droplets to limit evaporation thatmay occur during the melt/anneal steps. Droplets may be manipulatedusing a standard electrowetting process such as digital microfluidics(Fair, Microfluid Nanofluid (2007) 3: 245-281), or a light-inducedoptoelectrowetting (Chiou et al. Sensors and Actuators, A104 (2003)222-228). It should be appreciated that the electrowetting (oroptoelectrowetting) substrate (101) can be used as a general fluidicsmanipulator that couples to many different library substrates (102). Forexample, an instrument with one substrate (101) can be used to processmultiple DNA microarrays (102) in the same manner.

Some aspects of the invention relate to the transport of chargedmolecules such as nucleic acid (e.g. oligonucleotides orpolynucleotides) to a selected destination or selected feature on asupport within a fluid medium using a planar two dimensional path (x, yaxis). Preferably the molecules are electrophoretically transported bypolarization of the molecules of interest on application of a voltage,the charged molecule moving towards an electrode (anode or cathode). Insome embodiments, the array comprises one or more preferably, aplurality of electrophoretic planar microfluidic units, eachmicrofluidic unit comprising two electrodes. The electrodes systemcomprises at least one cathode and one anode. In some configurations,the cathode and anode are shared by a plurality of microfluidic units.In other configuration, the cathodes and anode are for a singlemicrofluidic unit. The microfluidic units enable the displacement ofcharged molecules of interest according to an electrophoretic path. Insome embodiments, each microfluidic unit comprises at least on channel.Preferably, each microfluidic unit is fluidly connected. For example,each microfluidic unit may be connected to another microfluidic unit, bya channel. In preferred embodiments, an aqueous buffer is utilized asthe fluid in the device. In some embodiments, each microfluidic unit maycomprise a capture site. In some embodiment, the capture sitecorresponds to an array feature. Yet in other embodiment, the capturesite corresponds to an array interfeature. In some embodiments, thecapture site comprises a material that capture charged molecules. Innucleic acids, the phosphate ion carries a negative charge. Accordingly,preferably the capture site comprises a material that capture negativelycharged molecules. In some instances, the capture material may capturethe charged molecules of interest by chemically interaction throughcovalent bonding, hydrogen bonding, ionic bonding, Vander Waalsinteractions, or other molecular interactions. Alternatively, thecapture material does not interact with the molecules of interest butretards the molecule's electrophoretic transport. In some embodiments,at least a first feature and a second feature of the arrays are in fluidcommunication and the charged oligonucleotide or polynucleotide is movedbetween the first feature and a second feature by applying a voltagebetween the first and the second feature.

In some aspects of the invention, the reaction volumes or a portion ofthe reaction volumes may be routed from a first substrate to a secondsubstrate, as illustrated in FIG. 26 and FIG. 27. In some embodiments, asubstrate (1901) can support individualized reaction volumes (1902) on asurface (1904), which can have surface properties designed to achieve aparticular contact angle between a reaction volume (1902) and thesurface (1904) (see FIG. 26). A second substrate (1903) can have anothersurface (1905) with properties designed to achieve other contactangle(s). The two surfaces (1904 and 1905) can be brought into closeproximity so that the reaction volume (1902 a) bridges both surfaces.Furthermore, the two substrates can then be pulled apart, breaking suchbridges (1902 a), and form two sets of separate volumes, one set calledsource volumes (1902 c) on the substrate surface (1904), and another setcalled destination volumes (1902 b) on the transferred surface (1905).The ratio of the source volume to destination volume is controlled bythe surface properties (e.g. contact angled) of the source surface(1904) and destination surface (1905). In another embodiment, thesubstrate (2001) can support individualized reaction volumes (2002),which can have surface properties designed to achieve a particularcontact angle between a reaction volume (2002) and the surface (2004). Asecond substrate (2003), designed to have features manufactured into thesubstrate forming surfaces (2005) that defines channels (2006). Thechannel forming surfaces (2005) can be designed with properties toachieve another particular contact angle. The channels can be aligned tothe reaction volumes (2002). The two substrates (2001 and 2003) can bebrought into close proximity so that the reaction volume (2002 a)bridges both substrates. The two substrates can then be pulled apart,breaking such bridges (2002 a), and forming two sets of separatevolumes, one set called source volumes (2002 c) on the substrate surface(2004), and another set called destination volumes (2002 b) on thetransferred surface (2005). The ratio of the source volume todestination volume is controlled by the surface properties of the sourcesurface (2004) and destination surface (2005).

In certain embodiments, the oligonucleotides are designed to provide thefull sense (plus strand) and antisense (minus strand) strands of thepolynucleotide construct. After hybridization of the plus and minusstrand oligonucleotides, double-stranded oligonucleotides are subjectedto ligation in order to form a first subassembly product. Subassemblyproducts are then subjected to ligation to form a larger nucleic acid orthe full nucleic acid sequence.

Ligase-based assembly techniques may involve one or more suitable ligaseenzymes that can catalyze the covalent linking of adjacent 3′ and 5′nucleic acid termini (e.g., a 5′ phosphate and a 3′ hydroxyl of nucleicacid(s) annealed on a complementary template nucleic acid such that the3′ terminus is immediately adjacent to the 5′ terminus). Accordingly, aligase may catalyze a ligation reaction between the 5′ phosphate of afirst nucleic acid to the 3′ hydroxyl of a second nucleic acid if thefirst and second nucleic acids are annealed next to each other on atemplate nucleic acid). A ligase may be obtained from recombinant ornatural sources. A ligase may be a heat-stable ligase. In someembodiments, a thermostable ligase from a thermophilic organism may beused. Examples of thermostable DNA ligases include, but are not limitedto: Tth DNA ligase (from Thermus thermophilus, available from, forexample, Eurogentec and GeneCraft); Pfu DNA ligase (a hyperthermophilicligase from Pyrococcus furiosus); Taq ligase (from Thermus aquaticus),any other suitable heat-stable ligase, or any combination thereof. Insome embodiments, one or more lower temperature ligases may be used(e.g., T4 DNA ligase). A lower temperature ligase may be useful forshorter overhangs (e.g., about 3, about 4, about 5, or about 6 baseoverhangs) that may not be stable at higher temperatures.

Non-enzymatic techniques can be used to ligate nucleic acids. Forexample, a 5′-end (e.g., the 5′ phosphate group) and a 3′-end (e.g., the3′ hydroxyl) of one or more nucleic acids may be covalently linkedtogether without using enzymes (e.g., without using a ligase). In someembodiments, non-enzymatic techniques may offer certain advantages overenzyme-based ligations. For example, non-enzymatic techniques may have ahigh tolerance of non-natural nucleotide analogues in nucleic acidsubstrates, may be used to ligate short nucleic acid substrates, may beused to ligate RNA substrates, and/or may be cheaper and/or more suitedto certain automated (e.g., high throughput) applications.

Non-enzymatic ligation may involve a chemical ligation. In someembodiments, nucleic acid termini of two or more different nucleic acidsmay be chemically ligated. In some embodiments, nucleic acid termini ofa single nucleic acid may be chemically ligated (e.g., to circularizethe nucleic acid). It should be appreciated that both strands at a firstdouble-stranded nucleic acid terminus may be chemically ligated to bothstrands at a second double-stranded nucleic acid terminus. However, insome embodiments only one strand of a first nucleic acid terminus may bechemically ligated to a single strand of a second nucleic acid terminus.For example, the 5′ end of one strand of a first nucleic acid terminusmay be ligated to the 3′ end of one strand of a second nucleic acidterminus without the ends of the complementary strands being chemicallyligated.

Accordingly, a chemical ligation may be used to form a covalent linkagebetween a 5′ terminus of a first nucleic acid end and a 3′ terminus of asecond nucleic acid end, wherein the first and second nucleic acid endsmay be ends of a single nucleic acid or ends of separate nucleic acids.In one aspect, chemical ligation may involve at least one nucleic acidsubstrate having a modified end (e.g., a modified 5′ and/or 3′ terminus)including one or more chemically reactive moieties that facilitate orpromote linkage formation. In some embodiments, chemical ligation occurswhen one or more nucleic acid termini are brought together in closeproximity (e.g., when the termini are brought together due to annealingbetween complementary nucleic acid sequences). Accordingly, annealingbetween complementary 3′ or 5′ overhangs (e.g., overhangs generated byrestriction enzyme cleavage of a double-stranded nucleic acid) orbetween any combination of complementary nucleic acids that results in a3′ terminus being brought into close proximity with a 5′ terminus (e.g.,the 3′ and 5′ termini are adjacent to each other when the nucleic acidsare annealed to a complementary template nucleic acid) may promote atemplate-directed chemical ligation. Examples of chemical reactions mayinclude, but are not limited to, condensation, reduction, and/orphoto-chemical ligation reactions. It should be appreciated that in someembodiments chemical ligation can be used to produce naturally occurringphosphodiester intemucleotide linkages, non-naturally-occurringphosphamide pyrophosphate internucleotide linkages, and/or othernon-naturally-occurring intemucleotide linkages.

In some embodiments, the process of chemical ligation may involve one ormore coupling agents to catalyze the ligation reaction. A coupling agentmay promote a ligation reaction between reactive groups in adjacentnucleic acids (e.g., between a 5′-reactive moiety and a 3′-reactivemoiety at adjacent sites along a complementary template). In someembodiments, a coupling agent may be a reducing reagent (e.g.,ferricyanide), a condensing reagent such (e.g., cyanoimidazole, cyanogenbromide, carbodiimide, etc.), or irradiation (e.g., UV irradiation forphoto-ligation).

In some embodiments, a chemical ligation may be an autoligation reactionthat does not involve a separate coupling agent. In autoligation, thepresence of a reactive group on one or more nucleic acids may besufficient to catalyze a chemical ligation between nucleic acid terminiwithout the addition of a coupling agent (see, for example, Xu et al.,(1997) Tetrahedron Lett. 38:5595-8). Non-limiting examples of thesereagent-free ligation reactions may involve nucleophilic displacementsof sulfur on bromoacetyl, tosyl, or iodo-nucleoside groups (see, forexample, Xu et al., (2001) Nat. Biotech. 19:148-52). Nucleic acidscontaining reactive groups suitable for autoligation can be prepareddirectly on automated synthesizers (see, for example, Xu et al., (1999)Nuc. Acids Res. 27:875-81). In some embodiments, a phosphorothioate at a3′ terminus may react with a leaving group (such as tosylate or iodide)on a thymidine at an adjacent 5′ terminus. In some embodiments, twonucleic acid strands bound at adjacent sites on a complementary targetstrand may undergo auto-ligation by displacement of a 5′-end iodidemoiety (or tosylate) with a 3′-end sulfur moiety. Accordingly, in someembodiments the product of an autoligation may include anon-naturally-occurring internucleotide linkage (e.g., a single oxygenatom may be replaced with a sulfur atom in the ligated product).

In some embodiments, a synthetic nucleic acid duplex can be assembledvia chemical ligation in a one step reaction involving simultaneouschemical ligation of nucleic acids on both strands of the duplex. Forexample, a mixture of 5′-phosphorylated oligonucleotides correspondingto both strands of a target nucleic acid may be chemically ligated by a)exposure to heat (e.g., to 97° C.) and slow cooling to form a complex ofannealed oligonucleotides, and b) exposure to cyanogen bromide or anyother suitable coupling agent under conditions sufficient to chemicallyligate adjacent 3′ and 5′ ends in the nucleic acid complex.

In some embodiments, a synthetic nucleic acid duplex can be assembledvia chemical ligation in a two step reaction involving separate chemicalligations for the complementary strands of the duplex. For example, eachstrand of a target nucleic acid may be ligated in a separate reactioncontaining phosphorylated oligonucleotides corresponding to the strandthat is to be ligated and non-phosphorylated oligonucleotidescorresponding to the complementary strand. The non-phosphorylatedoligonucleotides may serve as a template for the phosphorylatedoligonucleotides during a chemical ligation (e.g., using cyanogenbromide). The resulting single-stranded ligated nucleic acid may bepurified and annealed to a complementary ligated single-stranded nucleicacid to form the target duplex nucleic acid (see, for example, Shabarovaet al., (1991) Nucl. Acids Res. 19:4247-51).

In one aspect, a nucleic acid fragment may be assembled in a polymerasemediated assembly reaction from a plurality of oligonucleotides that arecombined and extended in one or more rounds of polymerase-mediatedextensions. In some embodiments, the oligonucleotides are overlappingoligonucleotides covering the full sequence but leaving single-strandedgaps that may be filed in by chain extension. The plurality of differentoligonucleotides may provide either positive sequences (plus strand),negative sequences (minus strand), or a combination of both positive andnegative sequences corresponding to the entire sequence of the nucleicacid fragment to be assembled. In some embodiments, one or moredifferent oligonucleotides may have overlapping sequence regions (e.g.,overlapping 5′ regions or overlapping 3′ regions). Overlapping sequenceregions may be identical (i.e., corresponding to the same strand of thenucleic acid fragment) or complementary (i.e., corresponding tocomplementary strands of the nucleic acid fragment). The plurality ofoligonucleotides may include one or more oligonucleotide pairs withoverlapping identical sequence regions, one or more oligonucleotidepairs with overlapping complementary sequence regions, or a combinationthereof. Overlapping sequences may be of any suitable length. Forexample, overlapping sequences may encompass the entire length of one ormore nucleic acids used in an assembly reaction. Overlapping sequencesmay be between about 5 and about 500 oligonucleotides long (e.g.,between about 10 and 100, between about 10 and 75, between about 10 and50, about 20, about 25, about 30, about 35, about 45, about 50, etc.).However, shorter, longer, or intermediate overlapping lengths may beused. It should be appreciated that overlaps between different inputnucleic acids used in an assembly reaction may have different lengths.

Polymerase-based assembly techniques may involve one or more suitablepolymerase enzymes that can catalyze a template-based extension of anucleic acid in a 5′ to 3′ direction in the presence of suitablenucleotides and an annealed template. A polymerase may be thermostable.A polymerase may be obtained from recombinant or natural sources. Insome embodiments, a thermostable polymerase from a thermophilic organismmay be used. In some embodiments, a polymerase may include a 3′ 5′exonuclease/proofreading activity. In some embodiments, a polymerase mayhave no, or little, proofreading activity (e.g., a polymerase may be arecombinant variant of a natural polymerase that has been modified toreduce its proofreading activity). Examples of thermostable DNApolymerases include, but are not limited to: Taq (a heat-stable DNApolymerase from the bacterium Thermus aquaticus); Pfu (a thermophilicDNA polymerase with a 3′→5′ exonuclease/proofreading activity fromPyrococcus furiosus, available from for example Promega); VentR® DNAPolymerase and VentRO (exo-) DNA Polymerase (thermophilic DNApolymerases with or without a 3′→5′ exonuclease/proofreading activityfrom Thermococcus litoralis; also known as Th polymerase); Deep VentR®DNA Polymerase and Deep VentR® (exo-) DNA Polymerase (thermophilic DNApolymerases with or without a 3′→5′ exonuclease/proofreading activityfrom Pyrococcus species GB-D; available from New England Biolabs); KODHiFi (a recombinant Thermococcus kodakaraensis KODI DNA polymerase witha 3′→5′ exonuclease/proofreading activity, available from Novagen);BIO-X-ACT (a mix of polymerases that possesses 5′-3′ DNA polymeraseactivity and 3′→5′ proofreading activity); Klenow Fragment (anN-terminal truncation of E. coli DNA Polymerase I which retainspolymerase activity, but has lost the 5′ 4 3′ exonuclease activity,available from, for example, Promega and NEB); Sequenase™ (T7 DNApolymerase deficient in T-5′ exonuclease activity); Phi29 (bacteriophage29 DNA polymerase, may be used for rolling circle amplification, forexample, in a TempliPhi™ DNA Sequencing Template Amplification Kit,available from Amersham Biosciences); TopoTaq (a hybrid polymerase thatcombines hyperstable DNA binding domains and the DNA unlinking activityof Methanopyrus topoisomerase, with no exonuclease activity, availablefrom Fidelity Systems); TopoTaq HiFi which incorporates a proofreadingdomain with exonuclease activity; Phusion™ (a Pyrococcus-like enzymewith a processivity-enhancing domain, available from New EnglandBiolabs); any other suitable DNA polymerase, or any combination of twoor more thereof.

In some embodiments, the polymerase can be a SDP (strand-displacingpolymerase; e.g, an SDPe—which is an SDP with no exonuclease activity).This allows isothermal PCR (isothermal extension, isothermalamplification) at a uniform temperature. As the polymerase (for example,Phi29, Bst) travels along a template it displaces the complementarystrand (e.g., created in previous extension reactions). As the displacedDNAs are single-stranded, primers can bind at a consistent temperature,removing the need for any thermocycling during amplification, therebyavoiding or decreasing evaporation of the reaction mixture.

It should be appreciated that the description of the assembly reactionsin the context of the oligonucleotides is not intended to be limiting.For example, other polynucleotides (e.g. single-stranded,double-stranded polynucleotides, restriction fragments, amplificationproducts, naturally occurring polynucleotides, etc.) may be included inan assembly reaction, along with one or more oligonucleotides, in orderto generate a polynucleotide of interest.

Aspects of the methods and devices provided herein may includeautomating one or more acts described herein. In some embodiments, oneor more steps of an amplification and/or assembly reaction may beautomated using one or more automated sample handling devices (e.g., oneor more automated liquid or fluid handling devices). Automated devicesand procedures may be used to deliver reaction reagents, including oneor more of the following: starting nucleic acids, buffers, enzymes(e.g., one or more ligases and/or polymerases), nucleotides, salts, andany other suitable agents such as stabilizing agents. Automated devicesand procedures also may be used to control the reaction conditions. Forexample, an automated thermal cycler may be used to control reactiontemperatures and any temperature cycles that may be used. In someembodiments, a scanning laser may be automated to provide one or morereaction temperatures or temperature cycles suitable for incubatingpolynucleotides. Similarly, subsequent analysis of assembledpolynucleotide products may be automated. For example, sequencing may beautomated using a sequencing device and automated sequencing protocols.Additional steps (e.g., amplification, cloning, etc.) also may beautomated using one or more appropriate devices and related protocols.It should be appreciated that one or more of the device or devicecomponents described herein may be combined in a system (e.g., a roboticsystem) or in a micro-environment (e.g., a micro-fluidic reactionchamber). Assembly reaction mixtures (e.g., liquid reaction samples) maybe transferred from one component of the system to another usingautomated devices and procedures (e.g., robotic manipulation and/ortransfer of samples and/or sample containers, including automatedpipetting devices, micro-systems, etc.). The system and any componentsthereof may be controlled by a control system.

Accordingly, method steps and/or aspects of the devices provided hereinmay be automated using, for example, a computer system (e.g., a computercontrolled system). A computer system on which aspects of the technologyprovided herein can be implemented may include a computer for any typeof processing (e.g., sequence analysis and/or automated device controlas described herein). However, it should be appreciated that certainprocessing steps may be provided by one or more of the automated devicesthat are part of the assembly system. In some embodiments, a computersystem may include two or more computers. For example, one computer maybe coupled, via a network, to a second computer. One computer mayperform sequence analysis. The second computer may control one or moreof the automated synthesis and assembly devices in the system. In otheraspects, additional computers may be included in the network to controlone or more of the analysis or processing acts. Each computer mayinclude a memory and processor. The computers can take any form, as theaspects of the technology provided herein are not limited to beingimplemented on any particular computer platform. Similarly, the networkcan take any form, including a private network or a public network(e.g., the Internet). Display devices can be associated with one or moreof the devices and computers. Alternatively, or in addition, a displaydevice may be located at a remote site and connected for displaying theoutput of an analysis in accordance with the technology provided herein.Connections between the different components of the system may be viawire, optical fiber, wireless transmission, satellite transmission, anyother suitable transmission, or any combination of two or more of theabove.

Each of the different aspects, embodiments, or acts of the technologyprovided herein can be independently automated and implemented in any ofnumerous ways. For example, each aspect, embodiment, or act can beindependently implemented using hardware, software or a combinationthereof. When implemented in software, the software code can be executedon any suitable processor or collection of processors, whether providedin a single computer or distributed among multiple computers. It shouldbe appreciated that any component or collection of components thatperform the functions described above can be generically considered asone or more controllers that control the above-discussed functions. Theone or more controllers can be implemented in numerous ways, such aswith dedicated hardware, or with general purpose hardware (e.g., one ormore processors) that is programmed using microcode or software toperform the functions recited above.

In this respect, it should be appreciated that one implementation of theembodiments of the technology provided herein comprises at least onecomputer-readable medium (e.g., a computer memory, a floppy disk, acompact disk, a tape, etc.) encoded with a computer program (i.e., aplurality of instructions), which, when executed on a processor,performs one or more of the above-discussed functions of the technologyprovided herein. The computer-readable medium can be transportable suchthat the program stored thereon can be loaded onto any computer systemresource to implement one or more functions of the technology providedherein. In addition, it should be appreciated that the reference to acomputer program which, when executed, performs the above-discussedfunctions, is not limited to an application program running on a hostcomputer. Rather, the term computer program is used herein in a genericsense to reference any type of computer code (e.g., software ormicrocode) that can be employed to program a processor to implement theabove-discussed aspects of the technology provided herein.

It should be appreciated that in accordance with several embodiments ofthe technology provided herein wherein processes are stored in acomputer readable medium, the computer implemented processes may, duringthe course of their execution, receive input manually (e.g., from auser).

Accordingly, overall system-level control of the assembly devices orcomponents described herein may be performed by a system controllerwhich may provide control signals to the associated nucleic acidsynthesizers, liquid handling devices, thermal cyclers, sequencingdevices, associated robotic components, as well as other suitablesystems for performing the desired input/output or other controlfunctions. Thus, the system controller along with any device controllerstogether form a controller that controls the operation of a nucleic acidassembly system. The controller may include a general purpose dataprocessing system, which can be a general purpose computer, or networkof general purpose computers, and other associated devices, includingcommunications devices, modems, and/or other circuitry or components toperform the desired input/output or other functions. The controller canalso be implemented, at least in part, as a single special purposeintegrated circuit (e.g., ASIC) or an array of ASICs, each having a mainor central processor section for overall, system-level control, andseparate sections dedicated to performing various different specificcomputations, functions and other processes under the control of thecentral processor section. The controller can also be implemented usinga plurality of separate dedicated programmable integrated or otherelectronic circuits or devices, e.g., hard wired electronic or logiccircuits such as discrete element circuits or programmable logicdevices. The controller can also include any other components ordevices, such as user input/output devices (monitors, displays,printers, a keyboard, a user pointing device, touch screen, or otheruser interface, etc.), data storage devices, drive motors, linkages,valve controllers, robotic devices, vacuum and other pumps, pressuresensors, detectors, power supplies, pulse sources, communication devicesor other electronic circuitry or components, and so on. The controlleralso may control operation of other portions of a system, such asautomated client order processing, quality control, packaging, shipping,billing, etc., to perform other suitable functions known in the art butnot described in detail herein.

Various aspects of the present invention may be used alone, incombination, or in a variety of arrangements not specifically discussedin the embodiments described in the foregoing and is therefore notlimited in its application to the details and arrangement of componentsset forth in the foregoing description or illustrated in the drawings.For example, aspects described in one embodiment may be combined in anymanner with aspects described in other embodiments.

Use of ordinal terms such as “first,” “second,” “third,” etc., in theclaims to modify a claim element does not by itself connote anypriority, precedence, or order of one claim element over another or thetemporal order in which acts of a method are performed, but are usedmerely as labels to distinguish one claim element having a certain namefrom another element having a same name (but for use of the ordinalterm) to distinguish the claim elements.

Also, the phraseology and terminology used herein is for the purpose ofdescription and should not be regarded as limiting. The use of“including,” “comprising,” or “having,” “containing,” “involving,” andvariations thereof herein, is meant to encompass the items listedthereafter and equivalents thereof as well as additional items.

EQUIVALENTS

The present invention provides among other things novel methods anddevices for high-fidelity gene assembly. While specific embodiments ofthe subject invention have been discussed, the above specification isillustrative and not restrictive. Many variations of the invention willbecome apparent to those skilled in the art upon review of thisspecification. The full scope of the invention should be determined byreference to the claims, along with their full scope of equivalents, andthe specification, along with such variations.

INCORPORATION BY REFERENCE

Reference is made to PCT application PCT/US09/55267, to U.S. provisionalapplication 61/257,591 filed Nov. 3, 2009, to U.S. Provisionalapplication 61/264,643, filed on Nov. 25, 2009, U.S. ProvisionalApplication 61/264,632, filed on Nov. 25, 2009, U.S. ProvisionalApplication 61/264,641 filed Nov. 25, 2009, U.S. Provisional Application61/293,192, filed Jan. 7, 2010, U.S. provisional application 61/310,100,filed on Mar. 3, 2010 and U.S. provisional application 61/310,100 filedMar. 3, 2010. All publications, patents and patent applications andsequence database entries mentioned herein are hereby incorporated byreference in their entirety as if each individual publication, patent orpatent application was specifically and individually indicated to beincorporated by reference.

What is claimed is:
 1. A method for preparing a plurality of oligonucleotides on a support, the method comprising: a. providing a support comprising a plurality of surface-bound single-stranded oligonucleotides contained within one or more droplets of a predefined volume of solution; b. exposing the plurality of surface-bound oligonucleotides to conditions suitable for a template-dependent synthesis reaction, thereby producing a plurality of complementary oligonucleotides; and c. adjusting or substantially maintaining the volume of the one or more droplets of solution.
 2. The method of claim 1 wherein the step of adjusting or substantially maintaining the volume of the one or more droplets of solution comprises maintaining the droplets under conditions that substantially limit solvent evaporation.
 3. The method of claim 1 wherein the plurality of surface-bound oligonucleotides are coupled to the surface at a feature that is selectively coated with a coating material.
 4. The method of claim 3 wherein the coating material has water trapping properties.
 5. The method of claim 4 wherein the coating material is selected from the group of colloidal silica, peptide gel, agarose, solgel and polydimethylsiloxane.
 6. The method of claim 2 wherein solvent evaporation is substantially limited by blocking the interface of the droplets with the atmosphere.
 7. The method of claim 6 wherein the droplets are overlaid with a non-miscible liquid thereby preventing water evaporation of the solution.
 8. The method of claim 7 wherein the non-miscible liquid forms a lipid bilayer.
 9. The method of claim 7 wherein the non-miscible liquid forms a thin film at the surface of the droplet.
 10. The method of claim 7 wherein the non-miscible liquid is a solvent.
 11. The method of claim 7 wherein the non-miscible liquid is mineral oil.
 12. The method of claim 7 wherein the non-miscible liquid is spotted onto the droplet.
 13. The method of claim 12 wherein the step of spotting is performed with an inkjet or mechanical device.
 14. The method of claim 1 further comprising monitoring the volume of the one or more droplets for evaporation.
 15. The method of claim 1 wherein the step of adjusting or substantially maintaining the volume of the one or more droplets of solution comprises adjusting droplet volume by providing additional solution in response to evaporation.
 16. The method of claim 15 wherein the step of adjusting droplet volume is performed with an inkjet device.
 17. The method of claim 2 wherein solvent evaporation is limited by increasing the humidity around the one or more droplets.
 18. The method of claim 17 wherein the humidity is increased locally by depositing satellite droplets in the vicinity of the one or more droplets.
 19. The method of claim 1 wherein the plurality of surface-bound oligonucleotides comprise a primer binding site, and wherein the solution comprises a polymerase, at least one primer and dNTPs and wherein the primer is complementary to the primer binding site.
 20. The method of claim 19 wherein the primer is a unique primer.
 21. The method of claim 19 wherein the primer is a universal primer.
 22. The method of claim 19 wherein the at least one primer is a pair of primer.
 23. The method of claim 22 wherein the pair of primers are unique primers.
 24. The method of claim 22 wherein the pair of primers are universal primers.
 25. The method of claim 19 wherein the plurality of surface-bound oligonucleotides is subjected to thermocycling thereby promoting primer extension within a droplet.
 26. The method of claim 25 further heating the surface to a denaturing temperature thereby providing a plurality of single-stranded complementary oligonucleotides within the one or more droplets.
 27. The method of claim 1 wherein the support comprises a plurality of discrete features, and wherein each feature comprises a plurality of surface-bound single-stranded oligonucleotides contained within a droplet of a predefined volume of solution.
 28. The method of claim 27 wherein each discrete feature comprises a plurality of surface-bound oligonucleotides having a different predefined sequence.
 29. The method for preparing a plurality of oligonucleotides, the method comprising the steps of: a. providing a support comprising a plurality of discrete features, each feature comprising a plurality of surface-bound single-stranded oligonucleotides having a predefined sequence, where each surface-bound oligonucleotide is hybridized to a synthesized oligonucleotide from a template-dependent reaction thereby forming a hybridized oligonucleotide duplex; b. heating the support to a first melting temperature under stringent melt conditions thereby denaturing the hybridized oligonucleotide duplexes comprising error-containing oligonucleotides and releasing error-containing oligonucleotides; c. removing the error-containing oligonucleotides from the solid support; d. denaturing error-free duplexes; and e. releasing error-free oligonucleotides in solution.
 30. The method of claim 29 wherein the stringent melt conditions are determined by a real-time melt curve.
 31. The method of claim 29 wherein the support is dehydrated and further comprising: hydrating at least one first feature of the support forming a droplet comprising hybridized oligonucleotides duplexes; and optionally hydrating at least a second feature of the support and repeating steps b-e on at least one second different feature and at least one different melting condition.
 32. The method of claim 29 wherein one or more discrete features are selectively heated.
 33. The method of claim 32 wherein the one or more discrete features are selectively heated using a digital mirror device.
 34. A method for generating on a support a plurality of single-stranded oligonucleotides for conducting a plurality of specified reactions within a droplet, the method comprising: a. providing a plurality of surface-bound single-stranded oligonucleotides wherein the oligonucleotides are suitable for hydration and wherein each oligonucleotides is bound to a discrete feature of the surface, each oligonucleotide having a predefined sequence different from the predefined sequence of the oligonucleotide bound to a different feature; b. selectively hydrating at least one predefined feature thereby providing hydrated oligonucleotides within at least one droplet; and c. exposing the hydrated oligonucleotides to further processing.
 35. A method of generating a selective set of amplified oligonucleotides of predefined sequences, the method comprising: a. providing a plurality of surface-bound single-stranded oligonucleotides wherein the plurality of oligonucleotides are suitable for hydration and wherein each plurality of oligonucleotides is bound to a discrete feature of the surface, wherein the predefined sequence of each plurality of oligonucleotides attached to the feature is different from the predefined sequence of the plurality of oligonucleotides attached to a different feature; b. selectively hydrating at least one selected feature thereby providing at least one plurality of hydrated oligonucleotides within a droplet; and c. amplifying the at least one plurality of hydrated oligonucleotides without amplifying the oligonucleotides at unselected features thereby generating at least one selective set of amplified oligonucleotides within the droplet.
 36. The method of claim 34 or claim 35 wherein the step of selectively hydrating comprises selectively spotting a solution promoting primer extension onto at least one feature creating at least one first stage droplet.
 37. The method of claim 36 wherein the solution comprises a polymerase, at least one primer and dNTPs wherein the primer is complementary to a primer binding site.
 38. The method of claim 37 wherein the primer is a unique primer.
 39. The method of claim 37 wherein the primer is a universal primer.
 40. The method of claim 37 wherein the at least one primer is a pair of primer.
 41. The method of claim 37 wherein the pair of primers are unique primers.
 42. The method of claim 37 wherein the pair of primers are universal primers.
 43. The method of claim 36 wherein at least one feature is subjected to thermocycling thereby promoting primer extension within a droplet.
 44. The method of claim 36 wherein the surface is subjected to thermocycling.
 45. The method of claim 43 or claim 44 wherein the thermocycling is modulated at the discrete hydrated features.
 46. The method of claim 34 further heating the surface to a denaturing temperature thereby providing a plurality of single-stranded complementary oligonucleotides within the at least one droplet.
 47. The method of claim 34 or claim 35 wherein the droplets are subjected to conditions limiting water evaporation.
 48. The method of claim 34 or claim 35 wherein the discrete features are selectively coated with a coating material.
 49. The method of claim 48 wherein the coating material has water trapping properties.
 50. The method of claim 49 wherein the coating material is selected from the group of colloidal silica, peptide gel, agarose, solgel and polydimethylsiloxane.
 51. The method of claim 47 wherein water evaporation is limited by blocking the interface of the droplet with the atmosphere.
 52. The method of claim 47 wherein the droplets are overlaid with a non-miscible liquid thereby preventing water evaporation of the solution.
 53. The method of claim 52 wherein the non-miscible liquid forms a lipid bilayer.
 54. The method of claim 52 wherein the non-miscible liquid forms a thin film at the surface of the droplet.
 55. The method of claim 52 wherein the non-miscible liquid is a solvent.
 56. The method of claim 52 wherein the non miscible liquid is mineral oil.
 57. The method of claim 52 wherein the non-miscible liquid is spotted onto the droplet.
 58. The method of claim 57 wherein the step of spotting is performed with an inkjet or mechanical device.
 59. The method of claim 47 further comprising adjusting droplet volume by addition solution to said droplet.
 60. The method of claim 59 wherein the addition is semi-continuous.
 61. The method of claim 59 wherein the step of adjusting droplet volume is performed with an inkjet device.
 62. The method of claim 47 wherein water evaporation is limited by controlling the humidity around the droplets.
 63. The method of claim 63 wherein the humidity is locally increased by depositing satellite droplets in the vicinity of the droplets.
 64. The method of claim 34 or claim 35 further comprising the step of removing error-containing oligonucleotides from a first plurality of amplified oligonucleotides, the method comprising the steps of: a. hydrating at least one first feature of the support following the amplification step forming a droplet comprising oligonucleotides duplexes; b. heating the surface to a first melting temperature under stringent melt conditions thereby denaturing duplexes comprising error-containing oligonucleotides and releasing error-containing oligonucleotides; c. removing the error-containing oligonucleotides from the surface; d. optionally repeating steps a through c on at least one second different feature and at least one different melting temperature; e. denaturing error-free duplexes; and f. releasing error-free oligonucleotides in solution.
 65. The method of claim 64 wherein the stringent melt conditions are determined by a real-time melt curve.
 66. The method of claim 64 wherein the surface is dried prior to step a.
 67. The method of claim 64 wherein one or more discrete features are selectively heated.
 68. The method of claim 67 wherein the one or more discrete features are selectively heated using a digital mirror device.
 69. A method for assembling at least one polynucleotide having a predefined sequence on a surface, the method comprising: a. providing a plurality of surface-bound single-stranded oligonucleotides having a predefined sequence wherein the plurality of oligonucleotides are suitable for hydration and wherein each plurality of oligonucleotides is bound to a discrete feature of the support, wherein the predefined sequence of each plurality of oligonucleotides attached to the feature is different from the predefined sequence of the plurality of oligonucleotides attached to a different feature; b. selectively hydrating at least one selected feature thereby providing hydrated oligonucleotides; c. synthesizing at least one plurality of oligonucleotides in a chain extension reaction on a first feature of the support by template-dependent synthesis; d. subjecting the products of chain extension to at least on round of denaturation and annealing; e. heating the support to a first melting temperature under stringent melt conditions thereby denaturing duplexes comprising error-containing oligonucleotides and releasing error-containing oligonucleotides in solution; f. removing the error-containing oligonucleotides from the surface; g. optionally repeating steps b-f on at least one second different feature and at least one different melting temperature; h. denaturing error-free duplexes; i. releasing error-free oligonucleotides in solution within a first stage droplet; j. combining a first droplet comprising a first plurality of substantially error-free oligonucleotides to a second droplet comprising a second plurality of substantially error-free oligonucleotides, wherein a terminal region of the second plurality of oligonucleotides comprises complementary sequences with a terminal region of the first plurality of oligonucleotides; and k. contacting the first and second plurality of oligonucleotides under conditions that allow one or more of annealing, chain extension and denaturing reaction.
 70. The method of claim 69 wherein the first and second droplets are combined by merging the droplets.
 71. The method of claim 69 wherein the step of combining comprises moving the droplet from a first feature to a second feature of the surface.
 72. The method of claim 71 wherein the droplets are moved using surface tension properties.
 73. The method of claim 69 wherein the surface is coated with a low melting-point substance for storage.
 74. The method of claim 73 wherein the low melting point substance is wax.
 75. The method of claim 69 wherein the reactions are initiated by heating the surface above the low-melting point.
 76. The method of claim 69 wherein the reactions are initiated by hydrating the discrete features.
 77. A method of moving a droplet on a substrate, the method comprising: a. providing a support surface comprising a plurality of modifiers, wherein the plurality of modifiers comprises a plurality of first modifiers and plurality of second modifiers, wherein the plurality of first modifiers has a contact angle smaller than the plurality of second modifiers and wherein the first and second modifiers partition the substrate according to a pattern; b. contacting a first modifier with a droplet, wherein the first modifier has a contact angle greater than the next first modifier; and c. moving the droplet on the surface along a desired path in the direction of the first modifiers having smaller contact angles.
 78. The method of claim 77 wherein each first modifier has a contact angle that is smaller than the previous one.
 79. The method of claim 77 wherein the plurality of first modifiers forms a hydrophilic gradient.
 80. The method of claim 78 wherein each first modifier has a contact angle that is at least 5° smaller than the previous one.
 81. The method of claim 77 wherein the difference in contact angle value between the first plurality of modifiers and the second plurality of modifiers is greater than 30°.
 82. The method of claim 77 wherein the first modifier comprises oligonucleotides.
 83. The method of claim 77 wherein the step of contacting is performed with an inkjet device.
 84. The method of claim 77 wherein the pattern is predetermined and wherein first modifiers alternate with second modifiers.
 85. The method of claim 84 wherein the first modifiers are surrounded with second modifiers.
 86. The method of claim 77 wherein the second modifier is the support surface.
 87. The method of claim 77 wherein the pattern forms a predetermined path along which the droplet moves.
 88. The method of claim 87 wherein the predetermined path is a hydrophilic gradient.
 89. A method of moving a droplet on support surface, the method comprising: a. providing a support surface comprising a plurality of features comprising a first modifier associated therewith, wherein the features are separated from each others by a second modifier and wherein the contact angle of the first modifier is different from the contact angle of the second modifier; b. contacting a first feature with a first droplet; c. contacting a second feature with a second droplet, wherein the second droplet volume is greater than the first droplet volume and wherein the second feature is adjacent to the first feature; d. contacting the first droplet with a third droplet; and e. merging the droplets into a fourth droplet, wherein the fourth droplet substantially covers the second feature surface thereby moving a first droplet from a first feature to a second feature using surface tension properties.
 90. A method of transferring droplets on support, the method comprising: a. providing a support comprising a plurality of addressable features, the features having different contact angles; b. contacting the support with a first droplet at a first feature; c. contacting the support with a second droplet at a second feature, wherein the second feature is adjacent to the first feature and the second feature contact angle is smaller than the first feature contact angle; d. contacting the first droplet with a third droplet; e. merging the first, second and third droplets into a fourth droplet, wherein the fourth droplet substantially covers the second feature thereby moving a first droplet along a desired path using surface tension directed properties.
 91. The method of claim 89 wherein the first modifier comprises oligonucleotides.
 92. The method of claim 89 wherein the contact angle of the second modifier is greater than the contact angle of the first modifier.
 93. The method of claim 89 wherein the first modifier is more hydrophilic than the second modifier.
 94. The method of claim 89 or claim 90 wherein the step of contacting is performed with an inkjet device.
 95. The method of claim 89 or claim 90 wherein the third droplet volume is smaller than the second droplet volume.
 96. The method of claim 89 wherein the difference in contact angles between the two modifiers is greater than 30°.
 97. The method of claim 89 or claim 90 wherein the fourth droplet volume comprises the first, second and third droplet volumes.
 98. The method of claim 89 or claim 90 further reducing the volume of the fourth droplet to the size of the first droplet volume and repeating steps c through e, thereby transferring the droplet along a desired path to a third feature by surface tension directed manipulation.
 99. A method for merging at least two droplets on a support, the method comprising: a. providing a support comprising a plurality of features; b. providing a first droplet on a first feature and second droplet on a second feature wherein the first and second features are adjacent; c. reducing the volume of the first droplet; d. moving the first droplet toward the second droplet; e. merging the first and the second droplet into a merged droplet; and f. optionally repeating c through e with a third droplet.
 100. The method of claim 99 wherein the first feature comprises a modifier having a contact angle greater than the modifier on the second feature.
 101. The method of claim 100 wherein each feature is separated from the other with a second modifier.
 102. A method of preparing a plurality of oligonucleotides, the method comprising: a. providing a first support comprising a plurality of discrete features, each feature comprising a plurality of surface-bound single-stranded oligonucleotides having a predefined sequence; b. providing a second support comprising an array of electrodes; c. providing at least one droplet on a first selected feature; d. synthesizing at least one plurality of oligonucleotides in a chain extension reaction on the first feature of the support by template-dependent synthesis; e. subjecting the products of chain extension to at least one round of denaturation and annealing to form duplex oligonucleotides; and d. exposing the duplexes to conditions promoting error reduction.
 103. The method of claim 102 wherein the droplet is moved to a second selected feature by activating and deactivating a selected set of electrodes.
 104. The method of claim 102 wherein the first and second support are the same.
 105. The method of claim 102 wherein the two supports are arranged together relative to each other by a distance sufficient to define a space between the two supports and wherein the droplet is located within the space.
 106. The method of claim 102 wherein the error reduction is an error filtration process.
 107. The method of claim 102 wherein the error reduction is an error correction process.
 108. The method of claim 102 wherein the error reduction is an error neutralization process.
 109. The method of claim 102 wherein error reduction utilizes a mismatch endonuclease.
 110. The method of claim 102 wherein the mismatch endonuclease is a CEL1 or a Surveyor™ endonuclease.
 111. The method of claim 110 wherein the mismatch endonuclease cleaves heteroduplexes.
 112. The method of claim 110 further comprising the steps of: a. exposing the error-containing duplexes with a mismatch endonuclease under conditions that permit cleavage of oligonucleotide duplexes having at least one mismatch; and b. removing cleaved duplexes.
 113. The method of claim 112 further comprising: a. denaturing surface-bound cleaved duplexes; b. removing single-stranded cleaved oligonucleotides; c. denaturing surface-bound substantially error free oligonucleotide duplexes; and d. releasing a first plurality of substantially error-free complementary oligonucleotides in a first droplet volume.
 114. The method of claim 112 further releasing a second plurality of substantially error-free oligonucleotides in a second droplet volume.
 115. The method of claim 112 further merging the first and second droplets.
 116. The method of claim 112 further activating and deactivating a set of electrodes to move the first and second droplet towards a third feature to form a merged droplet.
 117. The method of claim 112 further activating and deactivating a set of electrodes to move the first droplet towards the second droplet to form a merged droplet.
 118. The method of claim 115 wherein the step of forming a merged droplet mixes the first and second droplets composition together.
 119. The method of claim 114 further combining a first droplet comprising a first plurality of substantially error-free oligonucleotides to a second droplet comprising a second plurality of substantially error-free oligonucleotides, wherein a terminal region of the second plurality of oligonucleotides comprises complementary sequences with a terminal region of the first set of plurality of oligonucleotides; and contacting the first and second plurality of oligonucleotides under conditions that allow one or more of annealing, chain extension and denaturing reaction.
 120. The method of claim 102 wherein one or more discrete features are selectively heated.
 121. The method of claim 102 wherein the one or more discrete features are selectively heated using a digital mirror device.
 122. A method for preparing of a plurality of oligonucleotides having a predefined sequence on a support, the method comprising: a. providing a plurality of surface-bound single-stranded oligonucleotides having a predefined sequence wherein the plurality of oligonucleotides are suitable for hydration and wherein each plurality of oligonucleotides is bound to a discrete feature of the support, wherein the predefined sequence of each plurality of oligonucleotides attached to the feature is different from the predefined sequence of the plurality of oligonucleotides attached to a different feature; b. selectively inactivating at least one first feature by overlaying the first feature with an immiscible solution; c. selectively hydrating at least one second feature thereby providing hydrated oligonucleotides; d. synthesizing at least one plurality of oligonucleotides in a chain extension reaction on the second feature of the support by template-dependent synthesis; e. subjecting oligonucleotide duplexes to error-reduction; and f. releasing substantially error-free complementary oligonucleotides in a droplet volume.
 123. The method of claim 122 further activating an inactivated first feature by removing the immiscible solution.
 124. The method of claim 122 wherein the immiscible solution is oil.
 125. The method of claim 123 further a. selectively hydrating the first feature thereby providing hydrated oligonucleotides; b. synthesizing a plurality of oligonucleotides in a chain extension reaction on the first feature of the support by template-dependent synthesis; c. subjecting oligonucleotide duplexes to error-reduction; and d. releasing substantially error-free complementary oligonucleotides in a droplet volume.
 126. The method of claim 125 further moving the droplets to a third feature by electrowetting.
 127. The method of claim 1 wherein the plurality of single-stranded oligonucleotides are synthesized at each feature using high-voltage complementary semiconductor device.
 128. The method of claim 102 wherein the plurality of single-stranded oligonucleotides are synthesized at each feature using emulsion droplets.
 129. A method of synthesizing at least one oligonucleotide of a predefined sequence onto a support, the method comprising a. providing a first support comprising a plurality of discrete features; b. providing a second support comprising a high density array of electrodes; c. providing a droplet on a selected feature, the droplet comprising a reagent for performing a step of oligonucleotide synthesis; and d. moving the droplets using high voltage electronics to a second selected feature for performing a step of the oligonucleotide synthesis, thereby producing the oligonucleotide.
 130. A method of synthesizing at least one oligonucleotide of a predefined sequence onto a support, the method comprising a. providing a support comprising a plurality of discrete features; b. providing a first emulsion droplet on a selected feature, the droplet comprising a reagent for performing a step of oligonucleotide synthesis; and c. providing a second emulsion droplet onto the selected feature, the second droplet comprising a reagent for performing a step of oligonucleotide synthesis, thereby generating the oligonucleotide.
 131. The method of claim 130 further comprising a step of forming a merged droplet wherein the step of merging mixes a first and second droplets composition together.
 132. The method of claim 130 wherein each droplet comprises a reagent for the oligonucleotide synthesis, each reagent being encapsulated into an aqueous droplet within an immiscible compound.
 133. The method of claim 132 wherein the immiscible compound is an oil.
 134. The method of claim 129 or claim 130 wherein the reagents are selected from the group consisting of A coupling reagent, T coupling reagent, C coupling reagent, G coupling reagent, U coupling reagent, deblocking reagent, oxidation reagent, capping reagent.
 135. A method for monitoring a plurality of isolated reaction volumes on a support, the method comprising: a. providing a first support comprising a plurality isolated reaction volumes having a predefined surface-to-volume ratio; b. providing a second support comprising at least one monitoring isolated volume, wherein the monitoring volume has an identical surface-to-volume ratio to at least one of the reaction volume; and c. monitoring the volume of the at least one monitoring isolated volume, wherein the modification of the isolated monitoring volume is indicative of the modification of at least one isolated reaction volume.
 136. The method of claim 135 wherein the isolated volumes are droplets.
 137. The method of claim 135 wherein the isolated reaction volume comprises a solvent and wherein the monitoring volume comprises the same solvent.
 138. The method of claim 135 wherein the reaction volume comprises oligonucleotides.
 139. The method of claim 135 wherein the modification is an increase in volume.
 140. The method of claim 135 wherein the modification is a decrease in volume.
 141. The method of claim 135 wherein the first and second support are the same.
 142. The method of claim 135 wherein the isolated reaction volumes and the isolated monitoring volumes are placed on the same support.
 143. The method of claim 135 wherein the isolated reaction volumes and the isolated monitoring volumes are subjected to preselected conditions.
 144. The method of claim 143 wherein the preselected conditions include temperature, pressure, and gas mixture environment.
 145. The method of claim 144 wherein the surfaces of the isolated reaction volumes and the isolated monitoring volumes are in contact with the preselected gas mixture.
 146. The method of claim 145 wherein the gas mixture has a predefined molar ratio of solvent vapor and carrier gas.
 147. The method of claim 144 wherein the conditions are modified to induce isolated volume growth.
 148. The method of claim 144 wherein the conditions are modified to induce isolated volume evaporation.
 149. The method of claim 135 wherein the second support is a mirror.
 150. The method of claim 135 wherein the volume of the at least one monitoring isolated volume is monitored using an optical system.
 151. The method of claim 150 wherein the volume of the at least one monitoring isolated volume is monitored by measuring the intensity of an optical beam reflected on the second support.
 152. A method for monitoring a plurality of isolated reaction volumes on a support, the method comprising: a. providing a first support comprising a plurality isolated reaction volumes; b. providing a second support; and c. monitoring the condensation on the second support using an optical system.
 153. The method of claim 152 wherein the second support has a different surface tension property than the first support.
 154. The method of claim 153 wherein the second support is a mirror and wherein the condensation is monitored by measuring the intensity of an optical beam reflected on the mirror. 